Multiplex decoding of array sensors with microspheres

ABSTRACT

The invention relates to compositions and methods for multiplex decoding of microsphere array sensors.

This application claims the benefit of U.S. Ser. No. 60/302,213, filedJun. 28, 2001. This application claims the benefit of U.S. Ser. Nos.60/235,531, filed Sep. 26, 2000 and 60/172,106, filed Dec. 23, 1999, andis a continuing application of Ser. No. 09/344,526 filed Jun. 24, 1999now U.S. Pat. No. 7,060,431 and Ser. No. 09/189,543, filed Nov. 10,1998, which claims the benefit of 60/090,473, filed Jun. 24, 1998.

FIELD OF THE INVENTION

The invention relates to compositions and methods for decodingmicrosphere array sensors.

BACKGROUND OF THE INVENTION

There are a number of assays and sensors for the detection of thepresence and/or concentration of specific substances in fluids andgases. Many of these rely on specific ligand/antiligand reactions as themechanism of detection. That is, pairs of substances (i.e. the bindingpairs or ligand/antiligands) are known to bind to each other, whilebinding little or not at all to other substances. This has been thefocus of a number of techniques that utilize these binding pairs for thedetection of the complexes. These generally are done by labeling onecomponent of the complex in some way, so as to make the entire complexdetectable, using, for example, radioisotopes, fluorescent and otheroptically active molecules, enzymes, etc.

Of particular use in these sensors are detection mechanisms utilizingluminescence. Recently, the use of optical fibers and optical fiberstrands in combination with light absorbing dyes for chemical analyticaldeterminations has undergone rapid development, particularly within thelast decade. The use of optical fibers for such purposes and techniquesis described by Milanovich et al., “Novel Optical Fiber Techniques ForMedical Application”, Proceedings of the SPIE 28th Annual InternationalTechnical Symposium On Optics and Electro-Optics, Volume 494, 1980;Seitz, W. R., “Chemical Sensors Based On Immobilized Indicators andFiber Optics” in C.R.C. Critical Reviews In Analytical Chemistry, Vol.19, 1988, pp. 135–173; Wolfbeis, O. S., “Fiber Optical Fluorosensors InAnalytical Chemistry” in Molecular Luminescence Spectroscopy, Methodsand Applications (S. G. Schulman, editor), Wiley & Sons, New York(1988); Angel, S. M., Spectroscopy 2 (4):38 (1987); Walt, et al.,“Chemical Sensors and Microinstrumentation”, ACS Symposium Series, Vol.403, 1989, p. 252, and Wolfbeis, O. S., Fiber Optic Chemical Sensors,Ed. CRC Press, Boca Raton, Fla., 1991, 2nd Volume.

When using an optical fiber in an in vitro/in vivo sensor, one or morelight absorbing dyes are located near its distal end. Typically, lightfrom an appropriate source is used to illuminate the dyes through thefiber's proximal end. The light propagates along the length of theoptical fiber; and a portion of this propagated light exits the distalend and is absorbed by the dyes. The light absorbing dye may or may notbe immobilized; may or may not be directly attached to the optical fiberitself; may or may not be suspended in a fluid sample containing one ormore analytes of interest; and may or may not be retainable forsubsequent use in a second optical determination.

Once the light has been absorbed by the dye, some light of varyingwavelength and intensity returns, conveyed through either the same fiberor collection fiber(s) to a detection system where it is observed andmeasured. The interactions between the light conveyed by the opticalfiber and the properties of the light absorbing dye provide an opticalbasis for both qualitative and quantitative determinations.

Of the many different classes of light absorbing dyes whichconventionally are employed with bundles of fiber strands and opticalfibers for different analytical purposes are those more commoncompositions that emit light after absorption termed “fluorophores” andthose which absorb light and internally convert the absorbed light toheat, rather than emit it as light, termed “chromophores.”

Fluorescence is a physical phenomenon based upon the ability of somemolecules to absorb light (photons) at specified wavelengths and thenemit light of a longer wavelength and at a lower energy. Substances ableto fluoresce share a number of common characteristics: the ability toabsorb light energy at one wavelength_(ab); reach an excited energystate; and subsequently emit light at another light wavelength,_(em).The absorption and fluorescence emission spectra are individual for eachfluorophore and are often graphically represented as two separate curvesthat are slightly overlapping. The same fluorescence emission spectrumis generally observed irrespective of the wavelength of the excitinglight and, accordingly, the wavelength and energy of the exciting lightmay be varied within limits; but the light emitted by the fluorophorewill always provide the same emission spectrum. Finally, the strength ofthe fluorescence signal may be measured as the quantum yield of lightemitted. The fluorescence quantum yield is the ratio of the number ofphotons emitted in comparison to the number of photons initiallyabsorbed by the fluorophore. For more detailed information regardingeach of these characteristics, the following references are recommended:Lakowicz, J. R., Principles of Fluorescence Spectroscopy, Plenum Press,New York, 1983; Freifelder, D., Physical Biochemistry, second edition,W. H. Freeman and Company, New York, 1982; “Molecular LuminescenceSpectroscopy Methods and Applications: Part I” (S. G. Schulman, editor)in Chemical Analysis, vol. 77, Wiley & Sons, Inc., 1985; The Theory ofLuminescence, Stepanov and Gribkovskii, Iliffe Books, Ltd., London,1968.

In comparison, substances which absorb light and do not fluoresceusually convert the light into heat or kinetic energy. The ability tointernally convert the absorbed light identifies the dye as a“chromophore.” Dyes which absorb light energy as chromophores do so atindividual wavelengths of energy and are characterized by a distinctivemolar absorption coefficient at that wavelength. Chemical analysisemploying fiber optic strands and absorption spectroscopy using visibleand ultraviolet light wavelengths in combination with the absorptioncoefficient allow for the determination of concentration for specificanalyses of interest by spectral measurement. The most common use ofabsorbance measurement via optical fibers is to determine concentrationwhich is calculated in accordance with Beers' law; accordingly, at asingle absorbance wavelength, the greater the quantity of thecomposition which absorbs light energy at a given wavelength, thegreater the optical density for the sample. In this way, the totalquantity of light absorbed directly correlates with the quantity of thecomposition in the sample.

Many of the recent improvements employing optical fiber sensors in bothqualitative and quantitative analytical determinations concern thedesirability of depositing and/or immobilizing various light absorbingdyes at the distal end of the optical fiber. In this manner, a varietyof different optical fiber chemical sensors and methods have beenreported for specific analytical determinations and applications such aspH measurement, oxygen detection, and carbon dioxide analyses. Thesedevelopments are exemplified by the following publications: Freeman, etal., Anal Chem. 53:98 (1983); Lippitsch et al., Anal. Chem. Acta. 205:1,(1988); Wolfbeis et al., Anal. Chem. 60:2028 (1988); Jordan, et al.,Anal. Chem. 59:437 (1987); Lubbers et al., Sens. Actuators 1983;Munkholm et al., Talanta 35:109 (1988); Munkholm et al., Anal. Chem.58:1427 (1986); Seitz, W. R., Anal. Chem. 56:16A–34A (1984); Peterson,et al., Anal. Chem. 52:864 (1980): Saari, et al., Anal. Chem. 54:821(1982); Saari, et al., Anal. Chem. 55:667 (1983); Zhujun et al., Anal.Chem. Acta. 160:47 (1984); Schwab, et al., Anal. Chem. 56:2199 (1984);Wolfbeis, O. S., “Fiber Optic Chemical Sensors”, Ed. CRC Press, BocaRaton, Fla., 1991, 2nd Volume; and Pantano, P., Walt, D. R., Anal.Chem., 481A–487A, Vol. 67, (1995).

More recently, fiber optic sensors have been constructed that permit theuse of multiple dyes with a single, discrete fiber optic bundle. U.S.Pat. Nos. 5,244,636 and 5,250,264 to Walt, et al. disclose systems foraffixing multiple, different dyes on the distal end of the bundle, theteachings of each of these patents being incorporated herein by thisreference. The disclosed configurations enable separate optical fibersof the bundle to optically access individual dyes. This avoids theproblem of deconvolving the separate signals in the returning light fromeach dye, which arises when the signals from two or more dyes arecombined, each dye being sensitive to a different analyte, and there issignificant overlap in the dyes' emission spectra.

U.S. Ser. Nos. 08/818,199 and 09/151,877 describe array compositionsthat utilize microspheres or beads on a surface of a substrate, forexample on a terminal end of a fiber optic bundle, with each individualfiber comprising a bead containing an optical signature. Since the beadsgo down randomly, a unique optical signature is needed to “decode” thearray; i.e. after the array is made, a correlation of the location of anindividual site on the array with the bead or bioactive agent at thatparticular site can be made. This means that the beads may be randomlydistributed on the array, a fast and inexpensive process as compared toeither the in situ synthesis or spotting techniques of the prior art.Once the array is loaded with the beads, the array can be decoded, orcan be used, with full or partial decoding occurring after testing, asis more fully outlined below.

One drawback with the previous system is that it requires a set ofunique optical signatures. While large sets of such signatures areavailable, for example by using different ratios of different dyes, itwould be preferable to use decoding systems that do not rely on the useof sets of optical signatures. Accordingly, it is an object of theinvention to provide methods to allow decoding of bead arrays withoutrelying solely on unique optical signatures.

SUMMARY OF THE INVENTION

In accordance with the above objects, the present invention providesarray compositions comprising a substrate with a surface comprisingdiscrete sites. The composition further comprises a population ofmicrospheres comprising at least a first and a second subpopulation;each subpopulation comprises a bioactive agent; and an identifierbinding ligand that will bind a decoder binding ligand such that theidentity of the bioactive agent can be elucidated. The microspheres aredistributed on the surface.

In a further aspect, the present invention provides array compositionscomprising a substrate with a surface comprising discrete sites, and apopulation of microspheres comprising at least a first and a secondsubpopulation. Each subpopulation comprises a bioactive agent and doesnot comprise an optical signature.

In an additional aspect, the present invention provides methods ofmaking an array composition as outlined above. The methods compriseforming a surface comprising individual sites on a substrate anddistributing microspheres on said surface such that said individualsites contain microspheres. The microspheres comprise at least a firstand a second subpopulation each comprising a bioactive agent and do notcomprise an optical signature.

In a further aspect, the invention provides methods of making acomposition comprising forming a surface comprising individual sites ona substrate and distributing microspheres on the surface such that theindividual sites contain microspheres. The microspheres comprise atleast a first and a second subpopulation each comprising a bioactiveagent and an identifier binding ligand that will bind a decoder bindingligand such that the identification of the bioactive agent can beelucidated.

In an additional aspect, the invention provides methods of decoding anarray composition comprising providing an array composition as outlinedabove, and adding a plurality of decoding binding ligands to the arraycomposition to identify the location of at least a plurality of thebioactive agents.

In a further aspect, the invention provides methods of determining thepresence of a target analyte in a sample. The methods comprisecontacting the sample with an array composition as outlined herein, anddetermining the presence or absence of the target analyte.

In a further aspect, the invention provides a method comprisingproviding an array composition comprising a population of microspherescomprising at least a first and a second subpopulation, wherein eachsubpopulation comprises a bioactive agent and at least a first and asecond decoding attribute, and detecting each of said first and seconddecoding attributes to identify each of said bioactive agents.

In a further aspect the invention provides a method of increasing theinformation obtained in a decoding step. The method includes the use ofdegenerate probes as DBL-IBL combinations. In addition the inventionprovides the use of multiple decoding attributes on a bead.

In further aspect, the invention provides a method of increasing theconfidence of decoding. The method includes using the decoding as aquality control measure. In addition, the invention provides qualitycontrol fibers. In addition, the invention provides for parity analysisof decoding data.

In a further aspect, the invention provides a method of decoding anarray composition comprising providing an array composition comprising apopulation of microspheres comprising at least 50 subpopulations,wherein each subpopulation comprises a bioactive agent adding aplurality of decoding binding ligands to said population of microspheresto identify at least 50 of the bioactive agents.

In a further aspect, the invention provides a method of determining thepresence of a target analyte in a sample comprising contacting saidsample with a composition comprising a population of microspherescomprising at least 50 subpopulations, wherein each subpopulationcomprises a bioactive agent adding a plurality of decoding bindingligands to said population of microspheres to identify at least 50 ofthe bioactive agents and determining the presence or absence of saidtarget analyte.

FIGURES

FIG. 1 Illustrates a two color decoding process wherein eitherFAM-labeled or Cy3-labeled oligo complements are use to “paint” (label)the different bead types on the array.

FIG. 2 Depicts the decoding 128 different bead types with four colorsand four decode stages. (inset shows a single decode stage using fourdifferent dyes to decode 16 bead types.)

FIG. 3. Grey scale decoding of 16 different bead types. (A)Combinatorial pooling scheme for complementary decoding oligos. A (B)Two independent normalizing images were acquired, and the resulting beadintensities compared. (C) The alpha values (ratio of bead intensity inindicated decode stage to intensity in normalization image) are plottedfor three decodes stage described in (A).

FIG. 4 Depicts the use of fluorescence resonance energy transfer as acode for a probe on a random array. A. Depicts FRET oligos with linkersof different lengths. B. Depicts a plot of the ration of emission of thefluors from the oligos in FIG. 4A.

FIG. 5 Use of fluorescence resonance energy transfer to discriminatebetween linkers of varying length (SEQ ID NOS:1–6).

FIG. 6 depicts clustering in hyperspectral alpha space (Δ₁=I₁/6I_(I),Δ₂=I₂/6I_(i), Δ₃=I₃/6I_(i), etc.). A set of 128 different bead typespresent on a fiber bundle were decoded with by hybridizing set ofcomplementary oligonucleotides labeled with four dyes: Bodipy-493,Bodipy-R6G, Bodipy-TXR, and Bod-564 (only one dye per oligonucleotide).Shown is the second stage of a four stage decode in which 4013 beadswere decoded. Ovals are drawn around zones of hue clusters.

FIG. 7 depicts an error checking scheme.

FIG. 8 depicts an example of primer extension labeling of decoderoligonucleotides.

FIG. 9 depicts two color decoding of 16 beads with 16 distinct IBLs.

FIG. 10 depicts primer extension labeling of degenerate IBL-DBL probes.

FIG. 11 depicts a scatter plot of signals obtained from three distinctlabels recorded in two channels.

FIG. 12 depicts decoding by sequencing. A polymerase (e.g. Sequenase,Thermosequenase) and dye-labeled chain-terminating nucleotides are usedto sequence the encoding region of a DNA strand present on the bead. Thefour nucleotides are each labeled with a discrete label. Because only 4colors are detected at once, the colors are removed after each extensionstep. In subsequent extension steps, the primers are indexed or extendedby one base so that the primer can query the next base in the encodingregion.

FIG. 13 Depicts construction of probes on bead containing encodingsequences, zipcodes, and a gene-specific sequence. Two differentencoding cassettes are employed to facilitate the primer extensionreaction using primers with universal or degenerate bases. Using fourcolor sequencing and a single base code, 6 bases generates 4^6=4096codes, likewise four color hybridization using single hybridizationcolors also generates 4096 codes. The grand total number of codes is4096*4096=>16 million. If only a single color/two state scheme isemployed for hybridization, than 2^6=64 codes are generated. The grandtotal is 4096*64=262,144 codes. The zipcode sequences can also beconstructed so as to be overlapping to reduce the length of the overallsequence (i.e. cZip#1 vs. cZip#2).

FIG. 14 Depicts incorporation of a cleavable linker between encodingsequences and gene-specific sequence. A disulfide cleavable linker isshown for illustration purposes. If the linker is not present, theterminal portion of the gene-specific sequence can be included as partof the encoding sequence to reduce overall length of the probe.

FIG. 15 depicts decoding with exonuclease.

FIG. 16 depicts a graph showing decoding of about 1500 beadsubpopulation on each of several different fiber bundles.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is generally based on previous work comprising abead-based analytic chemistry system in which beads, also termedmicrospheres, carrying different chemical functionalities aredistributed on a substrate comprising a patterned surface of discretesites that can bind the individual microspheres. Since the beads aregenerally put onto the substrate randomly, the previous work relied onthe incorporation of unique optical signatures, generally fluorescentdyes, that could be used to identify the chemical functionality on anyparticular bead. This allows the synthesis of the candidate agents (i.e.compounds such as nucleic acids and antibodies) to be divorced fromtheir placement on an array, i.e. the candidate agents may besynthesized on the beads, and then the beads are randomly distributed ona patterned surface. Since the beads are first coded with an opticalsignature, this means that the array can later be “decoded”, i.e. afterthe array is made, a correlation of the location of an individual siteon the array with the bead or candidate agent at that particular sitecan be made. This means that the beads may be randomly distributed onthe array, a fast and inexpensive process as compared to either the insitu synthesis or spotting techniques of the prior art.

However, the drawback to these methods is that for a large array, thesystem requires a large number of different optical signatures, whichmay be difficult or time-consuming to utilize. Accordingly, the presentinvention provides several improvements over these methods, generallydirected to methods of coding and decoding the arrays. That is, as willbe appreciated by those in the art, the placement of the bioactiveagents is generally random, and thus a coding/decoding system isrequired to identify the bioactive agent at each location in the array.This may be done in a variety of ways, as is more fully outlined below,and generally includes: a) the use of decoding binding ligands (DBLs),generally directly labeled, that binds to either the bioactive agent orto identifier binding ligands (IBLs) attached to the beads; b)positional decoding, for example by either targeting the placement ofbeads (for example by using photoactivatible or photocleavable moietiesto allow the selective addition of beads to particular locations), or byusing either sub-bundles or selective loading of the sites, as are morefully outlined below; c) selective decoding, wherein only those beadsthat bind to a target are decoded; or d) combinations of any of these.In some cases, as is more fully outlined below, this decoding may occurfor all the beads, or only for those that bind a particular targetanalyte. Similarly, this may occur either prior to or after addition ofa target analyte.

Once the identity of the bioactive agent and its location in the arrayhas been fixed, the array is exposed to samples containing the targetanalytes, although as outlined below, this can be done prior to orduring the analysis as well. The target analytes will bind to thebioactive agents as is more fully outlined below, and result in a changein the optical signal of a particular bead.

In the present invention, “decoding” does not rely on the use of opticalsignatures, but rather on the use of decoding binding ligands that areadded during a decoding step. The decoding binding ligands will bindeither to a distinct identifier binding ligand partner that is placed onthe beads, or to the bioactive agent itself, for example when the beadscomprise single-stranded nucleic acids as the bioactive agents. Thedecoding binding ligands are either directly or indirectly labeled, andthus decoding occurs by detecting the presence of the label. By usingpools of decoding binding ligands in a sequential fashion, it ispossible to greatly minimize the number of required decoding steps.

In one embodiment, the invention provides methods for multiplex decodingof bead-arrays. That is, microspheres with bioactive agents are decodedto elucidate the identity of the bioactive agent on the bead. In apreferred embodiment at least 50 different bead types or subpopulationsare simultaneously decoded, or decoded in a single decoding experiment.More preferably at least 100; more preferably at least 500; morepreferably at least 1000; or preferably at least 1500 bead types aresimultaneously decoded or decoded in a single decoding experiment by themethods of the invention. Preferably at least 5000 bead types aresimultaneously decoded or decoded in a single decoding experiment.

Accordingly, the present invention provides array compositionscomprising at least a first substrate with a surface comprisingindividual sites. By “array” herein is meant a plurality of candidateagents in an array format; the size of the array will depend on thecomposition and end use of the array. Arrays containing from about 2different bioactive agents (i.e. different beads) to many millions canbe made, with very large fiber optic arrays being possible. Generally,the array will comprise from two to as many as a billion or more,depending on the size of the beads and the substrate, as well as the enduse of the array, thus very high density, high density, moderatedensity, low density and very low density arrays may be made. Preferredranges for very high density arrays are from about 10,000,000 to about2,000,000,000 (all numbers are per square cm), with from about100,000,000 to about 1,000,000,000 being preferred. High density arraysrange about 100,000 to about 10,000,000, with from about 1,000,000 toabout 5,000,000 being particularly preferred. Moderate density arraysrange from about 10,000 to about 100,000 being particularly preferred,and from about 20,000 to about 50,000 being especially preferred. Lowdensity arrays are generally less than 10,000, with from about 1,000 toabout 5,000 being preferred. Very low density arrays are less than1,000, with from about 10 to about 1000 being preferred, and from about100 to about 500 being particularly preferred. In some embodiments, thecompositions of the invention may not be in array format; that is, forsome embodiments, compositions comprising a single bioactive agent maybe made as well. In addition, in some arrays, multiple substrates may beused, either of different or identical compositions. Thus for example,large arrays may comprise a plurality of smaller substrates.

In addition, one advantage of the present compositions is thatparticularly through the use of fiber optic technology, extremely highdensity arrays can be made. Thus for example, because beads of 200 m orless (with beads of 200 nm possible) can be used, and very small fibersare known, it is possible to have as many as 40,000 or more (in someinstances, 1 million) different fibers and beads in a 1 mm² fiber opticbundle, with densities of greater than 15,000,000 individual beads andfibers (again, in some instances as many as 25–50 million) per 0.5 cm²obtainable.

By “substrate” or “solid support” or other grammatical equivalentsherein is meant any material that can be modified to contain discreteindividual sites appropriate for the attachment or association of beadsand is amenable to at least one detection method. As will be appreciatedby those in the art, the number of possible substrates is very large.Possible substrates include, but are not limited to, glass and modifiedor functionalized glass, plastics (including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, Teflon, etc.), polysaccharides, nylon ornitrocellulose, resins, silica or silica-based materials includingsilicon and modified silicon, carbon, metals, inorganic glasses,plastics, optical fiber bundles, and a variety of other polymers. Ingeneral, the substrates allow optical detection and do not themselvesappreciably fluoresce.

Generally the substrate is flat (planar), although as will beappreciated by those in the art, other configurations of substrates maybe used as well; for example, three dimensional configurations can beused, for example by embedding the beads in a porous block of plasticthat allows sample access to the beads and using a confocal microscopefor detection. Similarly, the beads may be placed on the inside surfaceof a tube, for flow-through sample analysis to minimize sample volume.Preferred substrates include optical fiber bundles as discussed below,and flat planar substrates such as glass, polystyrene and other plasticsand acrylics.

In a preferred embodiment, the substrate is an optical fiber bundle orarray, as is generally described in U.S. Ser. Nos. 08/944,850 and08/519,062, PCT US98/05025, and PCT US98/09163, all of which areexpressly incorporated herein by reference. Preferred embodimentsutilize preformed unitary fiber optic arrays. By “preformed unitaryfiber optic array” herein is meant an array of discrete individual fiberoptic strands that are co-axially disposed and joined along theirlengths. The fiber strands are generally individually clad. However, onething that distinguished a preformed unitary array from other fiberoptic formats is that the fibers are not individually physicallymanipulatable; that is, one strand generally cannot be physicallyseparated at any point along its length from another fiber strand.

Generally, the array of array compositions of the invention can beconfigured in several ways; see for example U.S. Ser. No. 09/473,904,and WO 00/39587, both of which are hereby expressly incorporated byreference. In a preferred embodiment, as is more fully outlined below, a“one component” system is used. That is, a first substrate comprising aplurality of assay locations (sometimes also referred to herein as“assay wells”), such as a microtiter plate, is configured such that eachassay location contains an individual array. That is, the assay locationand the array location are the same. For example, the plastic materialof the microtiter plate can be formed to contain a plurality of “beadwells” in the bottom of each of the assay wells. Beads containing thecapture probes of the invention can then be loaded into the bead wellsin each assay location as is more fully described below.

Alternatively, a “two component” system can be used. In this embodiment,the individual arrays are formed on a second substrate, which then canbe fitted or “dipped” into the first microtiter plate substrate. Apreferred embodiment utilizes fiber optic bundles as the individualarrays, generally with “bead wells” etched into one surface of eachindividual fiber, such that the beads containing the capture probes areloaded onto the end of the fiber optic bundle. The composite array thuscomprises a number of individual arrays that are configured to fitwithin the wells of a microtiter plate.

By “composite array” or “combination array” or grammatical equivalentsherein is meant a plurality of individual arrays, as outlined above.Generally the number of individual arrays is set by the size of themicrotiter plate used; thus, 96 well, 384 well and 1536 well microtiterplates utilize composite arrays comprising 96, 384 and 1536 individualarrays, although as will be appreciated by those in the art, not eachmicrotiter well need contain an individual array. It should be notedthat the composite arrays can comprise individual arrays that areidentical, similar or different. That is, in some embodiments, it may bedesirable to do the same 2,000 assays on 96 different samples;alternatively, doing 192,000 experiments on the same sample (i.e. thesame sample in each of the 96 wells) may be desirable. Alternatively,each row or column of the composite array could be the same, forredundancy/quality control. As will be appreciated by those in the art,there are a variety of ways to configure the system. In addition, therandom nature of the arrays may mean that the same population of beadsmay be added to two different surfaces, resulting in substantiallysimilar but perhaps not identical arrays.

At least one surface of the substrate is modified to contain discrete,individual sites for later association of microspheres. These sites maycomprise physically altered sites, i.e. physical configurations such aswells or small depressions in the substrate that can retain the beads,such that a microsphere can rest in the well, or the use of other forces(magnetic or compressive), or chemically altered or active sites, suchas chemically functionalized sites, electrostatically altered sites,hydrophobically/hydrophilically functionalized sites, spots of adhesive,etc.

The sites may be a pattern, i.e. a regular design or configuration, orrandomly distributed. A preferred embodiment utilizes a regular patternof sites such that the sites may be addressed in the X-Y coordinateplane. “Pattern” in this sense includes a repeating unit cell,preferably one that allows a high density of beads on the substrate.However, it should be noted that these sites may not be discrete sites.That is, it is possible to use a uniform surface of adhesive or chemicalfunctionalities, for example, that allows the association of beads atany position. That is, the surface of the substrate is modified to allowassociation of the microspheres at individual sites, whether or notthose sites are contiguous or non-contiguous with other sites. Thus, thesurface of the substrate may be modified such that discrete sites areformed that can only have a single associated bead, or alternatively,the surface of the substrate is modified and beads may go down anywhere,but they end up at discrete sites.

In a preferred embodiment, the surface of the substrate is modified tocontain wells, i.e. depressions in the surface of the substrate. Thismay be done as is generally known in the art using a variety oftechniques, including, but not limited to, photolithography, stampingtechniques, molding techniques and microetching techniques. As will beappreciated by those in the art, the technique used will depend on thecomposition and shape of the substrate.

In a preferred embodiment, physical alterations are made in a surface ofthe substrate to produce the sites. In a preferred embodiment, thesubstrate is a fiber optic bundle and the surface of the substrate is aterminal end of the fiber bundle, as is generally described in Ser. Nos.08/818,199 and 09/151,877, both of which are hereby expresslyincorporated by reference. In this embodiment, wells are made in aterminal or distal end of a fiber optic bundle comprising individualfibers. In this embodiment, the cores of the individual fibers areetched, with respect to the cladding, such that small wells ordepressions are formed at one end of the fibers. The required depth ofthe wells will depend on the size of the beads to be added to the wells.

Generally in this embodiment, the microspheres are non-covalentlyassociated in the wells, although the wells may additionally bechemically functionalized as is generally described below, cross-linkingagents may be used, or a physical barrier may be used, i.e. a film ormembrane over the beads.

In a preferred embodiment, the surface of the substrate is modified tocontain chemically modified sites, that can be used to associate, eithercovalently or non-covalently, the microspheres of the invention to thediscrete sites or locations on the substrate. “Chemically modifiedsites” in this context includes, but is not limited to, the addition ofa pattern of chemical functional groups including amino groups, carboxygroups, oxo groups and thiol groups, that can be used to covalentlyattach microspheres, which generally also contain corresponding reactivefunctional groups; the addition of a pattern of adhesive that can beused to bind the microspheres (either by prior chemicalfunctionalization for the addition of the adhesive or direct addition ofthe adhesive); the addition of a pattern of charged groups (similar tothe chemical functionalities) for the electrostatic association of themicrospheres, i.e. when the microspheres comprise charged groupsopposite to the sites; the addition of a pattern of chemical functionalgroups that renders the sites differentially hydrophobic or hydrophilic,such that the addition of similarly hydrophobic or hydrophilicmicrospheres under suitable experimental conditions will result inassociation of the microspheres to the sites on the basis ofhydroaffinity. For example, the use of hydrophobic sites withhydrophobic beads, in an aqueous system, drives the association of thebeads preferentially onto the sites. As outlined above, “pattern” inthis sense includes the use of a uniform treatment of the surface toallow association of the beads at discrete sites, as well as treatmentof the surface resulting in discrete sites. As will be appreciated bythose in the art, this may be accomplished in a variety of ways.

The compositions of the invention further comprise a population ofmicrospheres. By “population” herein is meant a plurality of beads asoutlined above for arrays. Within the population are separatesubpopulations, which can be a single microsphere or multiple identicalmicrospheres. That is, in some embodiments, as is more fully outlinedbelow, the array may contain only a single bead for each bioactiveagent; preferred embodiments utilize a plurality of beads of each type.

By “microspheres” or “beads” or “particles” or grammatical equivalentsherein is meant small discrete particles. The composition of the beadswill vary, depending on the class of bioactive agent and the method ofsynthesis. Suitable bead compositions include those used in peptide,nucleic acid and organic moiety synthesis, including, but not limitedto, plastics, ceramics, glass, polystyrene, methylstyrene, acrylicpolymers, paramagnetic materials, thoria sol, carbon graphite, titaniumdioxide, latex or cross-linked dextrans such as Sepharose, cellulose,nylon, cross-linked micelles and Teflon may all be used. “MicrosphereDetection Guide” from Bangs Laboratories, Fishers IN is a helpful guide.

The beads need not be spherical; irregular particles may be used. Inaddition, the beads may be porous, thus increasing the surface area ofthe bead available for either bioactive agent attachment or IBLattachment. The bead sizes range from nanometers, i.e. 100 nm, tomillimeters, i.e. 1 mm, with beads from about 0.2 micron to about 200microns being preferred, and from about 0.5 to about 5 micron beingparticularly preferred, although in some embodiments smaller beads maybe used.

It should be noted that a key component of the invention is the use of asubstrate/bead pairing that allows the association or attachment of thebeads at discrete sites on the surface of the substrate, such that thebeads do not move during the course of the assay.

Each microsphere comprises a bioactive agent, although as will beappreciated by those in the art, there may be some microspheres which donot contain a bioactive agent, depending on the synthetic methods. By“candidate bioactive agent” or“bioactive agent” or“chemicalfunctionality” or “binding ligand” herein is meant as used hereindescribes any molecule, e.g., protein, oligopeptide, small organicmolecule, coordination complex, polysaccharide, polynucleotide, etc.which can be attached to the microspheres of the invention. It should beunderstood that the compositions of the invention have two primary uses.In a preferred embodiment, as is more fully outlined below, thecompositions are used to detect the presence of a particular targetanalyte; for example, the presence or absence of a particular nucleotidesequence or a particular protein, such as an enzyme, an antibody or anantigen. In an alternate preferred embodiment, the compositions are usedto screen bioactive agents, i.e. drug candidates, for binding to aparticular target analyte.

Bioactive agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 Daltons.Bioactive agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The bioactiveagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Bioactive agents are also found amongbiomolecules including peptides, nucleic acids, saccharides, fattyacids, steroids, purines, pyrimidines, derivatives, structural analogsor combinations thereof. Particularly preferred are nucleic acids andproteins.

Bioactive agents can be obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means. Knownpharmacological agents may be subjected to directed or random chemicalmodifications, such as acylation, alkylation, esterification and/oramidification to produce structural analogs.

In a preferred embodiment, the bioactive agents are proteins. By“protein” herein is meant at least two covalently attached amino acids,which includes proteins, polypeptides, oligopeptides and peptides. Theprotein may be made up of naturally occurring amino acids and peptidebonds, or synthetic peptidomimetic structures. Thus “amino acid”, or“peptide residue”, as used herein means both naturally occurring andsynthetic amino acids. For example, homo-phenylalanine, citrulline andnorleucine are considered amino acids for the purposes of the invention.The side chains may be in either the (R) or the (S) configuration. Inthe preferred embodiment, the amino acids are in the (S) orL-configuration. If non-naturally occurring side chains are used,non-amino acid substituents may be used, for example to prevent orretard in vivo degradations.

In one preferred embodiment, the bioactive agents are naturallyoccurring proteins or fragments of naturally occuring proteins. Thus,for example, cellular extracts containing proteins, or random ordirected digests of proteinaceous cellular extracts, may be used. Inthis way libraries of procaryotic and eukaryotic proteins may be madefor screening in the systems described herein. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred.

In a preferred embodiment, the bioactive agents are peptides of fromabout 5 to about 30 amino acids, with from about 5 to about 20 aminoacids being preferred, and from about 7 to about 15 being particularlypreferred. The peptides may be digests of naturally occurring proteinsas is outlined above, random peptides, or “biased” random peptides. By“randomized” or grammatical equivalents herein is meant that eachnucleic acid and peptide consists of essentially random nucleotides andamino acids, respectively. Since generally these random peptides (ornucleic acids, discussed below) are chemically synthesized, they mayincorporate any nucleotide or amino acid at any position. The syntheticprocess can be designed to generate randomized proteins or nucleicacids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized bioactive proteinaceous agents.

In a preferred embodiment, a library of bioactive agents are used. Thelibrary should provide a sufficiently structurally diverse population ofbioactive agents to effect a probabilistically sufficient range ofbinding to target analytes. Accordingly, an interaction library must belarge enough so that at least one of its members will have a structurethat gives it affinity for the target analyte. Although it is difficultto gauge the required absolute size of an interaction library, natureprovides a hint with the immune response: a diversity of 10⁷–10⁸different antibodies provides at least one combination with sufficientaffinity to interact with most potential antigens faced by an organism.Published in vitro selection techniques have also shown that a librarysize of 10⁷ to 10⁸ is sufficient to find structures with affinity forthe target. Thus, in a preferred embodiment, at least 10⁶, preferably atleast 10⁷, more preferably at least 10⁸ and most preferably at least 10⁹different bioactive agents are simultaneously analyzed in the subjectmethods. Preferred methods maximize library size and diversity.

In a preferred embodiment, the library is fully randomized, with nosequence preferences or constants at any position. In a preferredembodiment, the library is biased. That is, some positions within thesequence are either held constant, or are selected from a limited numberof possibilities. For example, in a preferred embodiment, thenucleotides or amino acid residues are randomized within a definedclass, for example, of hydrophobic amino acids, hydrophilic residues,sterically biased (either small or large) residues, towards the creationof cysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

In a preferred embodiment, the bioactive agents are nucleic acids(generally called “probe nucleic acids” or “candidate probes” herein).By “nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramide (Beaucage, et al., Tetrahedron, 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem., 35:3800 (1970); Sprinzl,et al., Eur. J. Biochem., 81:579 (1977); Letsinger, et al., Nucl. AcidsRes., 14:3487 (1986); Sawai, et al., Chem. Lett., 805 (1984), Letsinger,et al., J. Am. Chem. Soc., 110:4470 (1988); and Pauwels, et al., ChemicaScripta, 26:141 (1986)), phosphorothioate (Mag, et al., Nucleic AcidsRes., 19:1437 (1991); and U.S. Pat. No. 5,644,048), phosphorodithioate(Briu, et al., J. Am. Chem. Soc., 111:2321 (1989)),O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press), and peptidenucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc.,114:1895 (1992); Meier, et al., Chem. Int. Ed. Engl., 31:1008 (1992);Nielsen, Nature, 365:566 (1993); Carlsson, et al., Nature, 380:207(1996), all of which are incorporated by reference)). Other analognucleic acids include those with positive backbones (Denpcy, et al.,Proc. Natl. Acad. Sci. USA, 92:6097 (1995)); non-ionic backbones (U.S.Pat. Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141; and 4,469,863;Kiedrowshi, et al., Angew. Chem. Intl. Ed. English, 30:423 (1991);Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988); Letsinger, etal., Nucleosides & Nucleotides, 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker, et al.,Bioorganic & Medicinal Chem. Lett., 4:395 (1994); Jeffs, et al., J.Biomolecular NMR, 34:17 (1994); Tetrahedron Lett., 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins,et al., Chem. Soc. Rev., (1995) pp. 169–176). Several nucleic acidanalogs are described in Rawls, C & E News, Jun. 2, 1997, page 35. Allof these references are hereby expressly incorporated by reference.These modifications of the ribose-phosphate backbone may be done tofacilitate the addition of additional moieties such as labels, or toincrease the stability and half-life of such molecules in physiologicalenvironments; for example, PNA is particularly preferred. In addition,mixtures of naturally occurring nucleic acids and analogs can be made.Alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made. Thenucleic acids may be single stranded or double stranded, as specified,or contain portions of both double stranded or single stranded sequence.The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid,where the nucleic acid contains any combination of deoxyribo- andribo-nucleotides, and any combination of bases, including uracil,adenine, thymine, cytosine, guanine, inosine, xanthanine,hypoxanthanine, isocytosine, isoguanine, and base analogs such asnitropyrrole and nitroindole, etc.

In a preferred embodiment, the bioactive agents are libraries of clonalnucleic acids, including DNA and RNA. In this embodiment, individualnucleic acids are prepared, generally using conventional methods(including, but not limited to, propagation in plasmid or phage vectors,amplification techniques including PCR, etc.). The nucleic acids arepreferably arrayed in some format, such as a microtiter plate format,and beads added for attachment of the libraries.

Attachment of the clonal libraries (or any of the nucleic acids outlinedherein) may be done in a variety of ways, as will be appreciated bythose in the art, including, but not limited to, chemical or affinitycapture (for example, including the incorporation of derivatizednucleotides such as AminoLink or biotinylated nucleotides that can thenbe used to attach the nucleic acid to a surface, as well as affinitycapture by hybridization), cross-linking, and electrostatic attachment,etc.

In a preferred embodiment, affinity capture is used to attach the clonalnucleic acids to the beads. For example, cloned nucleic acids can bederivatized, for example with one member of a binding pair, and thebeads derivatized with the other member of a binding pair. Suitablebinding pairs are as described herein for IBL/DBL pairs. For example,the cloned nucleic acids may be biotinylated (for example usingenzymatic incorporate of biotinylated nucleotides, for by photoactivatedcross-linking of biotin). Biotinylated nucleic acids can then becaptured on streptavidin-coated beads, as is known in the art.Similarly, other hapten-receptor combinations can be used, such asdigoxigenin and anti-digoxigenin antibodies. Alternatively, chemicalgroups can be added in the form of derivatized nucleotides, that canthem be used to add the nucleic acid to the surface.

Preferred attachments are covalent, although even relatively weakinteractions (i.e. non-covalent) can be sufficient to attach a nucleicacid to a surface, if there are multiple sites of attachment per eachnucleic acid. Thus, for example, electrostatic interactions can be usedfor attachment, for example by having beads carrying the opposite chargeto the bioactive agent.

Similarly, affinity capture utilizing hybridization can be used toattach cloned nucleic acids to beads. For example, as is known in theart, polyA+RNA is routinely captured by hybridization to oligo-dT beads;this may include oligo-dT capture followed by a cross-linking step, suchas psoralen crosslinking). If the nucleic acids of interest do notcontain a polyA tract, one can be attached by polymerization withterminal transferase, or via ligation of an oligoA linker, as is knownin the art.

Alternatively, chemical crosslinking may be done, for example byphotoactivated crosslinking of thymidine to reactive groups, as is knownin the art.

In general, special methods are required to decode clonal arrays, as ismore fully outlined below.

As described above generally for proteins, nucleic acid bioactive agentsmay be naturally occurring nucleic acids, random nucleic acids, or“biased” random nucleic acids. For example, digests of procaryotic oreukaryotic genomes may be used as is outlined above for proteins.

In general, probes of the present invention are designed to becomplementary to a target sequence (either the target analyte sequenceof the sample or to other probe sequences, as is described herein), suchthat hybridization of the target and the probes of the present inventionoccurs. This complementarily need not be perfect; there may be anynumber of base pair mismatches that will interfere with hybridizationbetween the target sequence and the single stranded nucleic acids of thepresent invention. However, if the number of mutations is so great thatno hybridization can occur under even the least stringent ofhybridization conditions, the sequence is not a complementary targetsequence. Thus, by “substantially complementary” herein is meant thatthe probes are sufficiently complementary to the target sequences tohybridize under the selected reaction conditions. High stringencyconditions are known in the art; see for example Maniatis et al.,Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and ShortProtocols in Molecular Biology, ed. Ausubel, et al., both of which arehereby incorporated by reference. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology—Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993). Generally, stringentconditions are selected to be about 5–10 C lower than the thermalmelting point (T_(m)) for the specific sequence at a defined ionicstrength pH. The T_(m) is the temperature (under defined ionic strength,pH and nucleic acid concentration) at which 50% of the probescomplementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30 Cfor short probes (e.g. 10 to 50 nucleotides) and at least about 60 C forlong probes (e.g. greater than 50 nucleotides). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. In another embodiment, less stringent hybridizationconditions are used; for example, moderate or low stringency conditionsmay be used, as are known in the art; see Maniatis and Ausubel, supra,and Tijssen, supra.

The term “target sequence”or grammatical equivalents herein means anucleic acid sequence on a single strand of nucleic acid. The targetsequence may be a portion of a gene, a regulatory sequence, genomic DNA,cDNA, RNA including mRNA and rRNA, or others. It may be any length, withthe understanding that longer sequences are more specific. As will beappreciated by those in the art, the complementary target sequence maytake many forms. For example, it may be contained within a largernucleic acid sequence, i.e. all or part of a gene or mRNA, a restrictionfragment of a plasmid or genomic DNA, among others. As is outlined morefully below, probes are made to hybridize to target sequences todetermine the presence or absence of the target sequence in a sample.Generally speaking, this term will be understood by those skilled in theart.

In a preferred embodiment, the bioactive agents are organic chemicalmoieties, a wide variety of which are available in the literature.

In a preferred embodiment, each bead comprises a single type ofbioactive agent, although a plurality of individual bioactive agents arepreferably attached to each bead. Similarly, preferred embodimentsutilize more than one microsphere containing a unique bioactive agent;that is, there is redundancy built into the system by the use ofsubpopulations of microspheres, each microsphere in the subpopulationcontaining the same bioactive agent.

As will be appreciated by those in the art, the bioactive agents mayeither be synthesized directly on the beads, or they may be made andthen attached after synthesis. In a preferred embodiment, linkers areused to attach the bioactive agents to the beads, to allow both goodattachment, sufficient flexibility to allow good interaction with thetarget molecule, and to avoid undesirable binding reactions.

In a preferred embodiment, the bioactive agents are synthesized directlyon the beads. As is known in the art, many classes of chemical compoundsare currently synthesized on solid supports, including beads, such aspeptides, organic moieties, and nucleic acids.

In a preferred embodiment, the bioactive agents are synthesized first,and then covalently attached to the beads. As will be appreciated bythose in the art, this will be done depending on the composition of thebioactive agents and the beads. The functionalization of solid supportsurfaces such as certain polymers with chemically reactive groups suchas thiols, amines, carboxyls, etc. is generally known in the art.Accordingly, “blank” microspheres may be used that have surfacechemistries that facilitate the attachment of the desired functionalityby the user. Some examples of these surface chemistries for blankmicrospheres include, but are not limited to, amino groups includingaliphatic and aromatic amines, carboxylic acids, aldehydes, amides,chloromethyl groups, hydrazide, hydroxyl groups, sulfonates andsulfates.

These functional groups can be used to add any number of differentcandidate agents to the beads, generally using known chemistries. Forexample, candidate agents containing carbohydrates may be attached to anamino-functionalized support; the aldehyde of the carbohydrate is madeusing standard techniques, and then the aldehyde is reacted with anamino group on the surface. In an alternative embodiment, a sulfhydryllinker may be used. There are a number of sulfhydryl reactive linkersknown in the art such as SPDP, maleimides, -haloacetyls, and pyridyldisulfides (see for example the 1994 Pierce Chemical Company catalog,technical section on cross-linkers, pages 155–200, incorporated hereinby reference) which can be used to attach cysteine containingproteinaceous agents to the support. Alternatively, an amino group onthe candidate agent may be used for attachment to an amino group on thesurface. For example, a large number of stable bifunctional groups arewell known in the art, including homobifunctional and heterobifunctionallinkers (see Pierce Catalog and Handbook, pages 155–200). In anadditional embodiment, carboxyl groups (either from the surface or fromthe candidate agent) may be derivatized using well known linkers (seethe Pierce catalog). For example, carbodiimides activate carboxyl groupsfor attack by good nucleophiles such as amines (see Torchilin et al.,Critical Rev. Therapeutic Drug Carrier Systems, 7(4):275–308 (1991),expressly incorporated herein). Proteinaceous candidate agents may alsobe attached using other techniques known in the art, for example for theattachment of antibodies to polymers; see Slinkin et al., Bioconi; Chem.2:342–348 (1991); Torchilin et al., supra; Trubetskoy et al., Bioconi;Chem. 3:323–327 (1992); King et al., Cancer Res. 54:6176–6185 (1994);and Wilbur et al., Bioconjugate Chem. 5:220–235 (1994), all of which arehereby expressly incorporated by reference). It should be understoodthat the candidate agents may be attached in a variety of ways,including those listed above. Preferably, the manner of attachment doesnot significantly alter the functionality of the candidate agent; thatis, the candidate agent should be attached in such a flexible manner asto allow its interaction with a target.

Specific techniques for immobilizing enzymes on microspheres are knownin the prior art. In one case, NH₂ surface chemistry microspheres areused. Surface activation is achieved with a 2.5% glutaraldehyde inphosphate buffered saline (10 mM) providing a pH of 6.9. (138 mM NaCl,2.7 mM, KCl). This is stirred on a stir bed for approximately 2 hours atroom temperature. The microspheres are then rinsed with ultrapure waterplus 0.01% tween 20 (surfactant) −0.02%, and rinsed again with a pH 7.7PBS plus 0.01% tween 20. Finally, the enzyme is added to the solution,preferably after being prefiltered using a 0.45 m amicon micropurefilter.

In some embodiments, the microspheres may additionally compriseidentifier binding ligands for use in certain decoding systems. By“identifier binding ligands” or “IBLs” herein is meant a compound thatwill specifically bind a corresponding decoder binding ligand (DBL) tofacilitate the elucidation of the identity of the bioactive agentattached to the bead. That is, the IBL and the corresponding DBL form abinding partner pair. By “specifically bind” herein is meant that theIBL binds its DBL with specificity sufficient to differentiate betweenthe corresponding DBL and other DBLs (that is, DBLs for other IBLs), orother components or contaminants of the system. The binding should besufficient to remain bound under the conditions of the decoding step,including wash steps to remove non-specific binding. In someembodiments, for example when the IBLs and corresponding DBLs areproteins or nucleic acids, the dissociation constants of the IBL to itsDBL will be less than about 10⁻⁴–10⁻⁶ M⁻¹, with less than about 10⁻⁵ to10⁻⁹ M⁻¹ being preferred and less than about 10⁻⁷–10⁻⁹ M⁻¹ beingparticulary preferred.

IBL-DBL binding pairs are known or can be readily found using knowntechniques. For example, when the IBL is a protein, the DBLs includeproteins (particularly including antibodies or fragments thereof (FAbs,etc.)) or small molecules, or vice versa (the IBL is an antibody and theDBL is a protein). Metal ion-metal ion ligands or chelators pairs arealso useful. Antigen-antibody pairs, enzymes and substrates orinhibitors, other protein-protein interacting pairs, receptor-ligands,complementary nucleic acids (including nucleic acid molecules that formtriple helices), and carbohydrates and their binding partners are alsosuitable binding pairs. Nucleic acid—nucleic acid binding proteins pairsare also useful, including single-stranded or double-stranded nucleicacid binding proteins, and small molecule nucleic acid binding agents.Similarly, as is generally described in U.S. Pat. Nos. 5,270,163,5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867, 5,705,337, andrelated patents, hereby incorporated by reference, nucleic acid“aptamers” can be developed for binding to virtually any target; such anaptamer-target pair can be used as the IBL-DBL pair. Similarly, there isa wide body of literature relating to the development of binding pairsbased on combinatorial chemistry methods.

In a preferred embodiment, the IBL is a protein, particularly an enzyme,that is conjugated to a bead. One advantage of using proteins/enzymes asIBLs is that many proteins and enzymes are commercially available inrelatively pure preparations. Proteins contain many suitable functionalgroups for attachment to beads. Moreover, the identity of many smallmolecules including substrates that bind the proteins/enzymes are known.Such small molecules serve as the DBL. In addition, the small moleculecan serve as the IBL and the enzyme as the DBL. As will be appreciatedby those in the art, combinations of enzymes and small molecules can beused as well, or multiple enzymes and small molecules. Another potentialadvantage relates to the purification of the labeled proteins. If oneconjugates a fluorophore to a recrystallizable protein, then theunlabeled protein can be removed from labeled protein by use ofcrystallization. By filtering off the crystalline solid, the labeledprotein will be concentrated in the filtrate.

In an alternative embodiment, antigen-antibody pairs are used as DBL-IBLcombinations. In one embodiment antibodies are conjugated to differentlabels, defined below, that are then used to decode antigen-labeledbeads.

In a preferred embodiment, the IBL is a molecule whose color orluminescence properties change in the presence of a selectively-bindingDBL.

In one embodiment, the DBL may be attached to a bead, i.e. a “decoderbead”, that may carry a label such as a fluorophore.

In a preferred embodiment, the IBL-DBL pair comprise substantiallycomplementary single-stranded nucleic acids. In this embodiment, thebinding ligands can be referred to as “identifier probes” and “decoderprobes”. Generally, the identifier and decoder probes range from about 4basepairs in length to about 1000, with from about 6 to about 100 beingpreferred, and from about 8 to about 40 being particularly preferred.What is important is that the probes are long enough to be specific,i.e. to distinguish between different IBL-DBL pairs, yet short enough toallow both a) dissociation, if necessary, under suitable experimentalconditions, and b) efficient hybridization.

In a preferred embodiment, as is more fully outlined below, the IBLs donot bind to DBLs. Rather, the IBLs are used as identifier moieties(“IMs”) that are identified directly, for example through the use ofmass spectroscopy.

Alternatively, in a preferred embodiment, the IBL and the bioactiveagent are the same moiety; thus, for example, as outlined herein,particularly when no optical signatures are used, the bioactive agentcan serve as both the identifier and the agent. For example, in the caseof nucleic acids, the bead-bound probe (which serves as the bioactiveagent) can also bind decoder probes, to identify the sequence of theprobe on the bead. Thus, in this embodiment, the DBLs bind to thebioactive agents. This is particularly useful as this embodiment cangive information about the array or the assay in addition to decoding.For example, as is more fully described below, the use of the DBLsallows array calibration and assay development. This may be done even ifthe DBLs are not used as such; for example in non-random arrays, the useof these probe sets can allow array calibration and assay developmenteven if decoding is not required.

In a preferred embodiment, the microspheres do not contain an opticalsignature. That is, as outlined in U.S. Ser. Nos. 08/818,199 and09/151,877, previous work had each subpopulation of microspherescomprising a unique optical signature or optical tag that is used toidentify the unique bioactive agent of that subpopulation ofmicrospheres; that is, decoding utilizes optical properties of the beadssuch that a bead comprising the unique optical signature may bedistinguished from beads at other locations with different opticalsignatures. Thus the previous work assigned each bioactive agent aunique optical signature such that any microspheres comprising thatbioactive agent are identifiable on the basis of the signature. Theseoptical signatures comprised dyes, usually chromophores or fluorophores,that were entrapped or attached to the beads themselves. Diversity ofoptical signatures utilized different fluorochromes, different ratios ofmixtures of fluorochromes, and different concentrations (intensities) offluorochromes.

Thus, the present invention does not rely solely on the use of opticalproperties to decode the arrays. However, as will be appreciated bythose in the art, it is possible in some embodiments to utilize opticalsignatures as an additional coding method, in conjunction with thepresent system. Thus, for example, as is more fully outlined below, thesize of the array may be effectively increased while using a single setof decoding moieties in several ways, one of which is the use incombination with optical signatures one beads. Thus, for example, usingone “set” of decoding molecules, the use of two populations of beads,one with an optical signature and one without, allows the effectivedoubling of the array size. The use of multiple optical signaturessimilarly increases the possible size of the array.

In a preferred embodiment, each subpopulation of beads comprises aplurality of different IBLs. By using a plurality of different IBLs toencode each bioactive agent, the number of possible unique codes issubstantially increased. That is, by using one unique IBL per bioactiveagent, the size of the array will be the number of unique IBLs (assumingno “reuse” occurs, as outlined below). However, by using a plurality ofdifferent IBLs per bead, n, the size of the array can be increased to2^(n), when the presence or absence of each IBL is used as theindicator. For example, the assignment of 10 IBLs per bead generates a10 bit binary code, where each bit can be designated as “1” (IBL ispresent) or “0” (IBL is absent). A 10 bit binary code has 2¹⁰ possiblevariants However, as is more fully discussed below, the size of thearray may be further increased if another parameter is included such asconcentration or intensity; thus for example, if two differentconcentrations of the IBL are used, then the array size increases as3^(n). Thus, in this embodiment, each individual bioactive agent in thearray is assigned a combination of IBLs, which can be added to the beadsprior to the addition of the bioactive agent, after, or during thesynthesis of the bioactive agent, i.e. simultaneous addition of IBLs andbioactive agent components.

Alternatively, when the bioactive agent is a polymer of differentresidues, i.e. when the bioactive agent is a protein or nucleic acid,the combination of different IBLs can be used to elucidate the sequenceof the protein or nucleic acid.

Thus, for example, using two different IBLs (IBL1 and IBL2), the firstposition of a nucleic acid can be elucidated: for example, adenosine canbe represented by the presence of both IBL1 and IBL2; thymidine can berepresented by the presence of IBL1 but not IBL2, cytosine can berepresented by the presence of IBL2 but not IBL1, and guanosine can berepresented by the absence of both. The second position of the nucleicacid can be done in a similar manner using IBL3 and IBL4; thus, thepresence of IBL1, IBL2, IBL3 and IBL4 gives a sequence of AA; IBL1,IBL2, and IBL3 shows the sequence AT; IBL1, IBL3 and IBL4 gives thesequence TA, etc. The third position utilizes IBL5 and IBL6, etc. Inthis way, the use of 20 different identifiers can yield a unique codefor every possible 10-mer.

The system is similar for proteins but requires a larger number ofdifferent IBLs to identify each position, depending on the alloweddiversity at each position. Thus for example, if every amino acid isallowed at every position, five different IBLs are required for eachposition. However, as outlined above, for example when using randompeptides as the bioactive agents, there may be bias built into thesystem; not all amino acids may be present at all positions, and somepositions may be preset; accordingly, it may be possible to utilize fourdifferent IBLs for each amino acid.

In this way, a sort of “bar code” for each sequence can be constructed;the presence or absence of each distinct IBL will allow theidentification of each bioactive agent.

In addition, the use of different concentrations or densities of IBLsallows a “reuse” of sorts. If, for example, the bead comprising a firstagent has a 1× concentration of IBL, and a second bead comprising asecond agent has a 10× concentration of IBL, using saturatingconcentrations of the corresponding labelled DBL allows the user todistinguish between the two beads.

In addition, the use of different ratios of labeled to unlabeled IBLsallows for a differentiation of the intensity rather than theconcentration of IBLs. For example, if a bead comprising a first agenthas a ratio of 1:1 (labeled: unlabeled IBL), while a second beadcomprising a second agent has a ration of 1:10 (labeled: unlabeled IBL),the user will distinguish the different intensities between the twobeads. This procedure offers the advantage that a broad spectrum ofintensities of labels can be created using only a single label.

Once the microspheres comprising the candidate agents and the uniqueIBLs are generated, they are added to the substrate to form an array. Itshould be noted that while most of the methods described herein add thebeads to the substrate prior to the assay, the order of making, usingand decoding the array can vary. For example, the array can be made,decoded, and then the assay done. Alternatively, the array can be made,used in an assay, and then decoded; this may find particular use whenonly a few beads need be decoded. Alternatively, the beads can be addedto the assay mixture, i.e. the sample containing the target analytes,prior to the addition of the beads to the substrate; after addition andassay, the array may be decoded. This is particularly preferred when thesample comprising the beads is agitated or mixed; this can increase theamount of target analyte bound to the beads per unit time, and thus (inthe case of nucleic acid assays) increase the hybridization kinetics.This may find particular use in cases where the concentration of targetanalyte in the sample is low; generally, for low concentrations, longbinding times must be used.

In addition, adding the beads to the assay mixture can allow sorting orselection. For example, a large library of beads may be added to asample, and only those beads that bind the sample may be added to thesubstrate. For example, if the target analyte is fluorescently labeled(either directly (for example by the incorporation of labels intonucleic acid amplification reactions) or indirectly (for example via theuse of sandwich assays)), beads that exhibit fluorescence as a result oftarget analyte binding can be sorted via Fluorescence Activated CellSorting (FACS) and only these beads added to an array and subsequentlydecoded. Similarly, the sorting may be accomplished through affinitytechniques; affinity columns comprising the target analytes can be made,and only those beads which bind are used on the array. Similarly, twobead systems can be used; for example, magnetic beads comprising thetarget analytes can be used to “pull out” those beads that will bind tothe targets, followed by subsequent release of the magnetic beads (forexample via temperature elevation) and addition to an array.

In general, the methods of making the arrays and of decoding the arraysis done to maximize the number of different candidate agents that can beuniquely encoded. The compositions of the invention may be made in avariety of ways. In general, the arrays are made by adding a solution orslurry comprising the beads to a surface containing the sites forassociation of the beads. This may be done in a variety of buffers,including aqueous and organic solvents, and mixtures. The solvent canevaporate, and excess beads removed.

In a preferred embodiment, when non-covalent methods are used toassociate the beads to the array, a novel method of loading the beadsonto the array is used. This method comprises exposing the array to asolution of particles (including microspheres and cells) and thenapplying energy, e.g. agitating or vibrating the mixture. This resultsin an array comprising more tightly associated particles, as theagitation is done with sufficient energy to cause weakly-associatedbeads to fall off (or out, in the case of wells). These sites are thenavailable to bind a different bead. In this way, beads that exhibit ahigh affinity for the sites are selected. Arrays made in this way havetwo main advantages as compared to a more static loading: first of all,a higher percentage of the sites can be filled easily, and secondly, thearrays thus loaded show a substantial decrease in bead loss duringassays. Thus, in a preferred embodiment, these methods are used togenerate arrays that have at least about 50% of the sites filled, withat least about 75% being preferred, and at least about 90% beingparticularly preferred. Similarly, arrays generated in this mannerpreferably lose less than about 20% of the beads during an assay, withless than about 10% being preferred and less than about 5% beingparticularly preferred.

In this embodiment, the substrate comprising the surface with thediscrete sites is immersed into a solution comprising the particles(beads, cells, etc.). The surface may comprise wells, as is describedherein, or other types of sites on a patterned surface such that thereis a differential affinity for the sites. This differnetial affinityresults in a competitive process, such that particles that willassociate more tightly are selected. Preferably, the entire surface tobe “loaded” with beads is in fluid contact with the solution. Thissolution is generally a slurry ranging from about 10,000:1beads:solution (vol:vol) to 1:1. Generally, the solution can compriseany number of reagents, including aqueous buffers, organic solvents,salts, other reagent components, etc. In addition, the solutionpreferably comprises an excess of beads; that is, there are more beadsthan sites on the array. Preferred embodiments utilize two-fold tobillion-fold excess of beads.

The immersion can mimic the assay conditions; for example, if the arrayis to be “dipped” from above into a microtiter plate comprising samples,this configuration can be repeated for the loading, thus minimizing thebeads that are likely to fall out due to gravity.

Once the surface has been immersed, the substrate, the solution, or bothare subjected to a competitive process, whereby the particles with loweraffinity can be disassociated from the substrate and replaced byparticles exhibiting a higher affinity to the site. This competitiveprocess is done by the introduction of energy, in the form of heat,sonication, stirring or mixing, vibrating or agitating the solution orsubstrate, or both.

A preferred embodiment utilizes agitation or vibration. In general, theamount of manipulation of the substrate is minimized to prevent damageto the array; thus, preferred embodiments utilize the agitation of thesolution rather than the array, although either will work. As will beappreciated by those in the art, this agitation can take on any numberof forms, with a preferred embodiment utilizing microtiter platescomprising bead solutions being agitated using microtiter plate shakers.

The agitation proceeds for a period of time sufficient to load the arrayto a desired fill. Depending on the size and concentration of the beadsand the size of the array, this time may range from about 1 second todays, with from about 1 minute to about 24 hours being preferred.

In a preferred embodiment, the substrate is pressed into a mixture ofdry beads (although slurries may be used as well), and then tapped toremove excess beads.

It should be noted that not all sites of an array may comprise a bead;that is, there may be some sites on the substrate surface which areempty. In addition, there may be some sites that contain more than onebead, although this is not preferred.

In some embodiments, for example when chemical attachment is done, it ispossible to associate the beads in a non-random or ordered way. Forexample, using photoactivatible attachment linkers or photoactivatibleadhesives or masks, selected sites on the array may be sequentiallyrendered suitable for attachment, such that defined populations of beadsare laid down.

The arrays of the present invention are constructed such thatinformation about the identity of the candidate agent is built into thearray, such that the random deposition of the beads in the fiber wellscan be “decoded” to allow identification of the candidate agent at allpositions. This may be done in a variety of ways, and either before,during or after the use of the array to detect target molecules.

Thus, after the array is made, it is “decoded” in order to identify thelocation of one or more of the bioactive agents, i.e. each subpopulationof beads, on the substrate surface.

In a preferred embodiment, a selective decoding system is used. In thiscase, only those microspheres exhibiting a change in the optical signalas a result of the binding of a target analyte are decoded. This iscommonly done when the number of “hits”, i.e. the number of sites todecode, is generally low. That is, the array is first scanned underexperimental conditions in the absence of the target analytes. Thesample containing the target analytes is added, and only those locationsexhibiting a change in the optical signal are decoded. For example, thebeads at either the positive or negative signal locations may be eitherselectively tagged or released from the array (for example through theuse of photocleavable linkers), and subsequently sorted or enriched in afluorescence-activated cell sorter (FACS). That is, either all thenegative beads are released, and then the positive beads are eitherreleased or analyzed in situ, or alternatively all the positives arereleased and analyzed. Alternatively, the labels may comprisehalogenated aromatic compounds, and detection of the label is done usingfor example gas chromatography, chemical tags, isotopic tags, or massspectral tags.

As will be appreciated by those in the art, this may also be done insystems where the array is not decoded; i.e. there need not ever be acorrelation of bead composition with location. In this embodiment, thebeads are loaded on the array, and the assay is run. The “positives”,i.e. those beads displaying a change in the optical signal as is morefully outlined below, are then “marked” to distinguish or separate themfrom the “negative” beads. This can be done in several ways, preferablyusing fiber optic arrays. In a preferred embodiment, each bead containsa fluorescent dye. After the assay and the identification of the“positives” or “active beads”, light is shown down either only thepositive fibers or only the negative fibers, generally in the presenceof a light-activated reagent (typically dissolved oxygen). In the formercase, all the active beads are photobleached. Thus, upon non-selectiverelease of all the beads with subsequent sorting, for example using afluorescence activated cell sorter (FACS) machine, the non-fluorescentactive beads can be sorted from the fluorescent negative beads.Alternatively, when light is shown down the negative fibers, all thenegatives are non-fluorescent and the the postives are fluorescent, andsorting can proceed. The characterization of the attached bioactiveagent may be done directly, for example using mass spectroscopy.

Alternatively, the identification may occur through the use ofidentifier moieties (“IMs”), which are similar to IBLs but need notnecessarily bind to DBLs. That is, rather than elucidate the structureof the bioactive agent directly, the composition of the IMs may serve asthe identifier. Thus, for example, a specific combination of IMs canserve to code the bead, and be used to identify the agent on the beadupon release from the bead followed by subsequent analysis, for exampleusing a gas chromatograph or mass spectroscope.

Alternatively, rather than having each bead contain a fluorescent dye,each bead comprises a non-fluorescent precursor to a fluorescent dye.For example, using photocleavable protecting groups, such as certainortho-nitrobenyl groups, on a fluorescent molecule, photoactivation ofthe fluorochrome can be done. After the assay, light is shown down againeither the “positive” or the “negative” fibers, to distinguish thesepopulations. The illuminated precursors are then chemically converted toa fluorescent dye. All the beads are then released from the array, withsorting, to form populations of fluorescent and non-fluorescent beads(either the positives and the negatives or vice versa).

In an alternate preferred embodiment, the sites of association of thebeads (for example the wells) include a photopolymerizable reagent, orthe photopolymerizable agent is added to the assembled array. After thetest assay is run, light is shown down again either the “positive” orthe “negative” fibers, to distinquish these populations. As a result ofthe irradiation, either all the positives or all the negatives arepolymerized and trapped or bound to the sites, while the otherpopulation of beads can be released from the array.

In a preferred embodiment, the location of every bioactive agent isdetermined using decoder binding ligands (DBLs). As outlined above, DBLsare binding ligands that will either bind to identifier binding ligands,if present, or to the bioactive agents themselves, preferably when thebioactive agent is a nucleic acid or protein.

In a preferred embodiment, as outlined above, the DBL binds to the IBL.

In a preferred embodiment, the bioactive agents are single-strandednucleic acids and the DBL is a substantially complementarysingle-stranded nucleic acid that binds (hybridizes) to the bioactiveagent, termed a decoder probe herein. A decoder probe that issubstantially complementary to each candidate probe is made and used todecode the array. In this embodiment, the candidate probes and thedecoder probes should be of sufficient length (and the decoding step rununder suitable conditions) to allow specificity; i.e. each candidateprobe binds to its corresponding decoder probe with sufficientspecificity to allow the distinction of each candidate probe.

In a preferred embodiment, the DBLs are either directly or indirectlylabeled. By “labeled” herein is meant that a compound has at least oneelement, isotope or chemical compound attached to enable the detectionof the compound. In general, labels fall into three classes: a) isotopiclabels, which may be radioactive or heavy isotopes; b) magnetic,electrical, thermal; and c) colored or luminescent dyes; although labelsinclude enzymes and particles such as magnetic particles as well.Examples of such lables include: peroxidase; alkaline phosphatase;biotin; urease; E alactosidase; BSA/KLH; gold particles; quantum dots;redox indicators; pH indicators; E-lactamase; luciferase; TSA; SPA;chemiluminescence; sonoluminescence; fluorophores; phosphors; and,fluorescent metal ion sensors. Preferred labels include luminescentlabels. In a preferred embodiment, the DBL is directly labeled, that is,the DBL comprises a label. In an alternate embodiment, the DBL isindirectly labeled; that is, a labeling binding ligand (LBL) that willbind to the DBL is used. In this embodiment, the labeling bindingligand-DBL pair can be as described above for IBL-DBL pairs. Suitablelabels include, but are not limited to, fluorescent lanthanidecomplexes, including those of Europium and Terbium, fluorescein,rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin,methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow,Cascade Blue™, Texas Red, FITC, PE, cy3, cy5 and others described in the6th Edition of the Molecular Probes Handbook by Richard P. Haugland,hereby expressly incorporated by reference. In addition, ion-sensitivedyes such as bis-BITC, Newport green, fluo 3, fura red find use in theinvention.

In one embodiment, the label is a molecule whose color or luminescenceproperties change in the presence of the IBL, due to a change in thelocal environment. For example, the label may be: (1) a fluorescent pHindicator whose emission intensity changes with pH; (2) a fluorescention indicator, whose emission properties change with ion concentration;or (3) a fluorescent molecule such as an ethidium salt whosefluorescence intensity increases in hydrophobic environments.

In one embodiment ion sensitive dyes are used for decoding. That is,dyes that fluoresce in the presence of certain ions are used. In apreferred embodiment the ion sensitive dyes are attached to the beads.An advantage of attaching dyes directly to microspheres is that the costassociated with oligonucleotide synthesis is avoided.

Accordingly, when such dyes are attached to the beads they can be usedto encode a specific nucleotide sequence, or the identity of thebioactive agent. Once the beads are distributed on the array, the arrayis contacted with buffers that include certain ions. In the presence ofappropriate ion, certain dyes will fluoresce and fluorescence isdetected. In a preferred embodiment, the array is contacted withdifferent buffers sequentially. Each buffer contains a different ion.Preferably the array is washed between buffer solutions so that anysignal produced as a result of the contact with the ion solution isdiminished.

In this embodiment multiple different ion sensitive dyes can be used.That is, preferably, more than 2 with more than 3 or 4 dyes are attachedto the microspheres. In some embodiments, more than 10 dyes are attachedto the microsphere.

One advantage to using ion sensitive dyes is that fluorescencebleed-through is diminished. That is, because the response of the dyesis either positive or negative, i.e. they interact or they don't,bleed-through is avoided.

In a preferred embodiment, decoding of self-assembled random arrays isdone on the basis of pH titration. In this embodiment, in addition tobioactive agents, the beads comprise optical signatures, wherein theoptical signatures are generated by the use of pH-responsive dyes(sometimes referred to herein as “pH dyes”) such as fluorophores. Thisembodiment is similar to that outlined in PCT US98/05025 and U.S. Ser.No. 09/151,877, both of which are expressly incorporated by reference,except that the dyes used in the present invention exhibits changes influorescence intensity (or other properties) when the solution pH isadjusted from below the pKa to above the pKa (or vice versa). In apreferred embodiment, a set of pH dyes is used, each with a differentpKa, preferably separated by at least 0.5 pH units. Preferredembodiments utilize a pH dye set of pka's of 2.0, 2.5, 3.0, 3.5, 4.0,4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11,and 11.5. Each bead can contain any subset of the pH dyes, and in thisway a unique code for the bioactive agent is generated. Thus, thedecoding of an array is achieved by titrating the array from pH 1 to pH13, and measuring the fluorescence signal from each bead as a functionof solution pH.

In one embodiment, the decoder probes are labeled in situ; that is, theyneed not be labeled prior to the decoding reaction. In this embodiment,the incoming decoder probe is shorter than the candidate probe, creatinga 5′ “overhang” on the decoding probe. The addition of labeled ddNTPs(each labeled with a unique tag) and a polymerase will allow theaddition of the tags in a sequence specific manner, thus creating asequence-specific pattern of signals. Similarly, other modifications canbe done, including ligation, etc.

In one embodiment, a series of sequential extension and denaturingstages allows for positional decoding of the beads. That is, followingone round of extension, the DBL is removed or stripped from the IBL anda second round of extension is performed. The contents of any givenprimer extension reaction is shown in FIG. 8.

In the primer extension method, up to four fluorescently-labeleddideoxynucleotide terminators each with a different colored label (e.g.using ABI's dRhodamine® terminators such as dR110, dR6G, dTAMRA, dROX orBig Dye® Terminators) could be used for ‘painting’. The number of uniquebeads that can be decoded is given by N=y^(x), where n=the number ofunique codes, y=the number of colors and x=the number of stages orprimer extension reactions. This formula yields 4096 unique codes using6 primer extension stages and four colors (i.e. 4096=4⁶).

In a preferred embodiment the bioactive agent is synthesized withencoding sequences that allows decoding to be accomplished independentof the useable portion of the bioactive agent, i.e. nucleic acidsequence. By “useable” portion is meant the adapter sequence or targetsequence. In a preferred embodiment the bioactive agent also issynthesized with a primer region to facilitate sequencing or primerextension analysis of the encoding sequence (see FIG. 12). As shown inFIG. 12, a primer complementary to a portion of the ssDNA on the bead isextended by single base polymerase extension using dye-labeled chainterminating nucleotides. After incorporation of a labeled, i.e.fluorescent, chain terminator and reading of the array, i.e. detectingthe label, the fluorescence is removed. Removal of the fluorescence isaccomplished directly when reversible chain terminators are used (seeCanard and Sarfati, Gene, 148(1): 1–6, 1994, which is expresslyincorporated herein by reference). However, in a preferred embodimentthe primer is dissociated from the bead and a subsequent primer isannealed. Preferably the subsequent primer is 1 nucleotide longer thanthe former primer so that an additional nucleotide in the encodingsequence can be interrogated. In a preferred embodiment the subsequentprimer is lengthened by the addition of a degenerate base. This can beaccomplished either by incorporating all 4 nucleotides into theadditional position of the primer, or by the incorporation of auniversal base into the primer. The new primer is then subject toextension as described above. The primer extension can be repeated asmany times as are necessary to decode the microsphere.

Decoding by sequencing has the advantage that encoding a vast number ofdistinct codes by using only a small number of nucleotides. That is, theinformation content of the encoding region depends on the number ofcombinations of the bases used, i.e. 4 when A, C, G and T are used, thatare present at each position and the length of the encoding region. When4 nucleotides are used, there are up to fifteen possible combinations ofnucleotides at each position (A, C, G, T, A/C, A/G, A/T, C/G, C/T, G/T,A/C/G, A/C/T, A/G/T, C/G/G and A/C/G/T). As such, the informationcontent is 15^(n), where n is equal to the length of the encodingsequence. As such, for example, with just 3 base positions, 3375different codes can be generated.

In addition an additional preferred embodiment includes the combinationof decoding by sequencing with decoding by hybridization. In oneapplication of decoding by sequencing, universal primers, each one baselonger than the previous, are sequentially hybridized to the array. Thebase adjacent to the 3′ end of the universal primer is determined by asingle base extension reaction employing labeled-chain terminators,preferably using four different color labels one for each base. Since ahybridization event must be performed for each base read, once canimprove the decoding power by using the hybridization event to alsodecode. However, rather than using universal primers, zipcode sequenceswould be used instead (see FIG. 13)

The placement of the encoding sequences can be either on the “bead-side”of the gene-specific sequence or on the terminal side. Placement of theencoding sequences on the terminal side offers that advantage ofincorporating a cleavable linker between the encoding sequences and thegene-specific sequence (FIG. 14). After completion of decoding, theencoding sequences can be removed to prevent possible interference inthe analytical assay.

In one embodiment following decoding, the label is extinguished. By“extinguished” is meant that the signal from the label is eliminated.This is accomplished by a variety of techniques including cleaving thelabel or destroying the signal of the label. That is, in one embodiment,the label is attached to the DBL via a cleavable linker. Following adecoding step, the linker is cleaved thereby releasing the label. In oneembodiment cleavage of the label results in deprotection of thebioactive agent, for example when the bioactive agent contains aprotecting group. Alternatively, cleavage of the label preserves thenature of the bioactive agent. That is, what is important is thatcleavage of the label does not interfere with the bioactive agent.

In an alternative embodiment, the label is not cleaved, but rather iseliminated by destroying the signal of the label. That is, followingdecoding, the label is contacted with an agent that degrades or destroysthe signal. For example, organic fluorescent dyes are subject tochemical degradation under appropriate conditions. Thus, following adecoding step where the fluorescent signal of the dyes are detected, thefluorophore is destroyed by an appropriate chemical solution thatdegrades the fluorescent molecule sufficient leaving no trace offluorescence. In this way, beads are tagged with fluorescent labels,i.e. barcodes of sorts, their location or identity deciphered andrecorded. The beads are then exposed to the “fluorescent destroyingsolution” and the fluorescence is eliminated, allowing an analyticalassay to be conducted. Suitable fluorescent dyes include but are notlimited to various bodipy, or Alexa dyes from Molecular Probes. The“fluorescent destroying solution” is a reducing or oxidizing solution,such as 0.01 SM sodium periodate.

In a preferred embodiment not only is the label cleaved, but theencoding nucleic acid is cleaved. This method finds particular use inremoving residual signal following a decoding step. Accordingly, in apreferred embodiment a cleavable linker can be used to bridge theadapter and beads. After decoding, the adapter oligonucleotide(s) can beeasily removed through cleavable linkers. There are many commerciallyavailable cleavable linkers. They are routinely used in thecombinatorial solid-phase synthesis field. The linkers can be cleavedunder acid, base or photo radiation conditions.

In addition, restriction enzymes can be used to cleave a linker.Alternatively, because of the sequence specificity of restrictionenzymes, they also are used to cleave encoding nucleic acids. A changein signal following incubation with a sequence specific enzyme decodesthe bead. An example is illustrative.

Consider the decoding of 6 bead types with three zip codes or adapters.In general, the strategy will have B=(2^n)−2. Where B is the number oftypes encoded and n is the number of zip codes. The bead types are z1,z2, z3, z1z2, z1z3, and z2z3. Here zizj indicates that both codes are ona bead. Every code is already dye labeled.

Generally,each code is designed with a site that can be specifically cutwith some agent. This includes, but is not limited to, restrictionenzymes. For ease of description, the process using restriction enzymesand zip codes that contain specific restriction sites is described. Whenrestriction enzymes are used, the zip codes are double stranded. Eachstage would consist of sequentially adding one restriction enzyme. Animage is taken prior to stage 1, and the intensity of every bead ismeasured. Restriction enzyme 1 (Re1) is added. New intensitymeasurements are taken. The information of which beads decrease inintensity, and which remain the same. Detecting the pattern of beads forwhich the signal “decreased” and “stayed the same” uniquely determinesthe bead type. If we denote “decreased” by 1 and “stayed the same” by 0,then the decoding would give the following codes:

-   z1: 1,0,0-   z2: 0,1,0-   z3: 0,0,1-   z1z2: 1,1,0-   z1z3: 1,0,1-   z2z3: 0,1,1.

The general approach uses n codes, which would give (2^n)−2 types.Subtracting 2 leaves off the all off state, and the all zip codes usedstate.

In another embodiment the array is decoded by using exonuclease enzyme.In this embodiment the microspheres are encoded with dye-labelednucleotides. Preferably each nucleotide is labeled with a discretelabel. In this particular embodiment it is desirable to protect thebioactive agent from cleavage. This can be accomplished by a variety ofknown methods including hybridizing the bioactive agent to acomplementary sequence or blocking the terminus of the bioactive agent.Alternatively, if the exonuclease enzyme operates from 5′ to 3′, one candesign zipcodes from 5′ to 3′ and SNP-gene specific oligos from 3′ to5′. This way zipcode sequences will be operated on by the exonuclease,but the SNP/gene-specific sequences will remain intact.

The microsphere is then contacted with an exonuclease that successivelyremoves the labeled nucleotides of the encoding sequence. Monitoring ofthe signal of the microspheres should begin upon addition of theexonuclease and monitored in all channels that correspond to the signalemitted by the labels in use. For example, assume the microsphere hasall the zipcodes available (with the above specifications), and is in asolution. At time T=0, the exonuclease enzyme is applied to thesolution. At the same time, the acquisition system (CCD) starts takingimages every few seconds (FIG. 15). The assumption is that the reactionof the exonuclease is slow enough, so one would have ample time to takeseveral images, as the reaction is proceeding. If this is not the case,the reaction can be slowed down by increasing the stringency conditions.

The exonuclease removes the nucleotides, one at a time. Therefore,ideally, one would notice a step reduction in the signal of theappropriate channel, once the nucleotide is removed (and moved away fromthe focal zone). The complete set of waveforms (i.e., the four colorchannels, and from T0 to Tend) will provide a unique signature for everyzipcode.

In practice, the removal of the nucleotides will not be completelysynchronized on different oligos (as shown in FIG. 15). For instance,for two oligos on the same bead (same zipcode), the exonuclease may haveremoved one nucleotide from one, and two nucleotides from the other.However, since one monitors many oligos from each bead, the overalltrend should be preserved, i.e., in general the nucleotides closer tothe end should be removed first. This means that in general, althoughthe transitions on the waveforms may not be sharp, the signatures couldstill be unique.

Accordingly, the identification of the location of the individual beads(or subpopulations of beads) is done using one or more decoding stepscomprising a binding between the labeled DBL and either the IBL or thebioactive agent (i.e. a hybridization between the candidate probe(sometimes referred to herein as a “decoding sequence) and the decoderprobe when the bioactive agent is a nucleic acid). After decoding, theDBLs can be removed and the array can be used; however, in somecircumstances, for example when the DBL binds to an IBL and not to thebioactive agent, the removal of the DBL is not required (although it maybe desirable in some circumstances). In addition, as outlined herein,decoding may be done either before the array is used in an assay, duringthe assay, or after the assay.

In one embodiment, a single decoding step is done. In this embodiment,each DBL is labeled with a unique label, such that the the number ofunique labels is equal to or greater than the number of bioactive agents(although in some cases, “reuse” of the unique labels can be done, asdescribed herein; similarly, minor variants of candidate probes canshare the same decoder, if the variants are encoded in anotherdimension, i.e. in the bead size or label). For each bioactive agent orIBL, a DBL is made that will specifically bind to it and contains aunique label, for example one or more fluorochromes. Thus, the identityof each DBL, both its composition (i.e. its sequence when it is anucleic acid) and its label, is known. Then, by adding the DBLs to thearray containing the bioactive agents under conditions which allow theformation of complexes (termed hybridization complexes when thecomponents are nucleic acids) between the DBLs and either the bioactiveagents or the IBLs, the location of each DBL can be elucidated. Thisallows the identification of the location of each bioactive agent; therandom array has been decoded. The DBLs can then be removed, ifnecessary, and the target sample applied.

In a preferred embodiment, the number of unique labels is less than thenumber of unique bioactive agents, and thus a sequential series ofdecoding steps are used. To facilitate the discussion, this embodimentis explained for nucleic acids, although other types of bioactive agentsand DBLs are useful as well. In this embodiment, decoder probes aredivided into n sets for decoding. The number of sets corresponds to thenumber of unique tags. Each decoder probe is labeled in n separatereactions with n distinct tags. All the decoder probes share the same ntags. Each pool of decoders contains only one of the n tag versions ofeach decoder, and no two decoder probes have the same sequence of tagsacross all the pools. The number of pools required for this to be trueis determined by the number of decoder probes and the n. Hybridizationof each pool to the array generates a signal at every address comprisingan IBL. The sequential hybridization of each pool in turn will generatea unique, sequence-specific code for each candidate probe. Thisidentifies the candidate probe at each address in the array. Forexample, if four tags are used, then 4×n sequential hybridizations canideally distinguish 4^(n) sequences, although in some cases more stepsmay be required. After the hybridization of each pool, the hybrids aredenatured and the decoder probes removed, so that the probes arerendered single-stranded for the next hybridization (although it is alsopossible to hybridize limiting amounts of target so that the availableprobe is not saturated. Sequential hybridizations can be carried out andanalyzed by subtracting pre-existing signal from the previoushybridization).

An example is illustrative. Assuming an array of 16 probe nucleic acids(numbers 1–16), and four unique tags (four different fluors, forexample; labels A–D). Decoder probes 1–16 are made that correspond tothe probes on the beads. The first step is to label decoder probes 1–4with tag A, decoder probes 5–8 with tag B, decoder probes 9–12 with tagC, and decoder probes 13–16 with tag D. The probes are mixed and thepool is contacted with the array containing the beads with the attachedcandidate probes. The location of each tag (and thus each decoder andcandidate probe pair) is then determined. The first set of decoderprobes are then removed. A second set is added, but this time, decoderprobes 1, 5, 9 and 13 are labeled with tag A, decoder probes 2, 6, 10and 14 are labeled with tag B, decoder probes 3, 7, 11 and 15 arelabeled with tag C, and decoder probes 4, 8, 12 and 16 are labeled withtag D. Thus, those beads that contained tag A in both decoding stepscontain candidate probe 1; tag A in the first decoding step and tag B inthe second decoding step contain candidate probe 2; tag A in the firstdecoding step and tag C in the second step contain candidate probe 3;etc. As will be appreciated by those in the art, the decoder probes canbe made in any order and added in any order.

In addition, since the size of the array will be set by the number ofunique decoding binding ligands, there are a number of techniques thatallow new combinations and the “reuse” of DBLs to allow for a greaternumber of test sites. This may be done in a wide variety of ways, asoutlined below.

In a preferred embodiment, a spatial or positional coding system isdone. In this embodiment, there are sub-bundles or subarrays (i.e.portions of the total array) that are utilized. By analogy with thetelephone system, each subarray is an “area code”, that can have thesame labels (i.e. telephone numbers) of other subarrays, that areseparated by virtue of the location of the subarray. Thus, for example,the same unique labels can be reused from bundle to bundle. Thus, theuse of 50 unique labels in combination with 100 different subarrays canform an array of 5000 different bioactive agents. In this embodiment, itbecomes important to be able to identify one bundle from another; ingeneral, this is done either manually or through the use of markerbeads; these can be beads containing unique tags for each subarray, orthe use of the same marker bead in differing amounts, or the use of twoor more marker beads in different ratios.

In a preferred embodiment, the coding and decoding is accomplished bysequential loading of the microspheres into the array. As outlined abovefor spatial coding, in this embodiment, the optical signatures can be“reused”. In this embodiment, the library of microspheres eachcomprising a different bioactive agent (or the subpopulations eachcomprise a different bioactive agent), is divided into a plurality ofsublibraries; for example, depending on the size of the desired arrayand the number of unique tags, 10 sublibraries each comprising roughly10% of the total library may be made, with each sublibrary comprisingroughly the same unique tags. Then, the first sublibrary is added to thefiber optic bundle comprising the wells, and the location of eachbioactive agent is determined, generally through the use of DBLs. Thesecond sublibrary is then added, and the location of each bioactiveagent is again determined. The signal in this case will comprise thesignal from the “first” DBL and the “second” DBL; by comparing the twomatrices the location of each bead in each sublibrary can be determined.Similarly, adding the third, fourth, etc. sublibraries sequentially willallow the array to be filled.

In a preferred embodiment, codes can be “shared” in several ways. In afirst embodiment, a single code (i.e. IBL/DBL pair) can be assigned totwo or more agents if the target analytes different sufficiently intheir binding strengths. For example, two nucleic acid probes used in anmRNA quantitation assay can share the same code if the ranges of theirhybridization signal intensities do not overlap. This can occur, forexample, when one of the target sequences is always present at a muchhigher concentration than the other. Alternatively, the two targetsequences might always be present at a similar concentration, but differin hybridization efficiency.

Alternatively, a single code can be assigned to multiple agents if theagents are functionally equivalent. For example, if a set ofoligonucleotide probes are designed with the common purpose of detectingthe presence of a particular gene, then the probes are functionallyequivalent, even though they may differ in sequence. Similarly, ifclasses or “families” of analytes are desired, all probes for differentmembers of a class such as kinases or G-protein coupled receptors couldshare a code. Similarly, an array of this type could be used to detecthomologs of known genes. In this embodiment, each gene is represented bya heterologous set of probes, hybridizing to different regions of thegene (and therefore differing in sequence). The set of probes share acommon code. If a homolog is present, it might hybridize to some but notall of the probes. The level of homology might be indicated by thefraction of probes hybridizing, as well as the average hybridizationintensity. Similarly, multiple antibodies to the same protein could allshare the same code.

In a preferred embodiment, the set of codes can be increased using akind of degeneracy on the IBLs on the beads. For most of the systemsdescribed herein, particularly when the IBL is a nucleic acid (includingthe case where the nucleic acid IBL is also the candidate agent), anysingle bead contains a single IBL sequence (decoding sequence). However,in this embodiment, it is possible to have a single bead comprise two ormore decoding sequences, that differ at a particular nucleotideposition. For a degeneracy of two at position X, there are 10 differentpossible combinations: the decoding sequence has an A at position X, aT, a C, a G, or a mixture of decoding sequences with either A/T, N/G,A/C, C/G, C/T or G/T. By using decoder probes that have different labelsfor each different nucleotide at the position, the number of codes canbe increased, since beads containing the two different sequences willexhibit a mixture of signals. In addition, as will be appreciated bythose in the art, rather than a “degeneracy” of two, degeneracies ofthree or four can be done as well. Thus, a bead containing threedifferent decoding sequences will exhibit a signal comprising threedifferent colors, etc.

Decoding the degenerate probes is done by a series of hybridizations tolabeled oligonucleotides. Each hybridization decodes one position in theprobe so the total number of hybridizations needed to decode a libraryis equal to the length of the variable region. Each hybridization isdone with four oligonucleotides, each designed to hybridize specificallyto a sequence containing a specific base at the interrogated positionand each tagged with a distinctly colored fluorescent dye.

In a preferred embodiment, a degenerate probe sequence is attached toone or more constant probe regions. By “constant” probe region is meanta portion of a probe that is invariant among a population of decoderprobes. In a particularly preferred embodiment, the degenerate orvariable region is flanked on either side by constant regions. As such,each oligonucletoide has the following structure:

Although the C1 and C2 regions may be any length, preferably each isfrom 5 to 50 nucleotides in length. More preferably each is from 7 to 30nucleotides in length and most preferably, each is about 10 bp inlength. In one embodiment, the constant regions do not vary in sequenceamong the probes designed to decode a particular set of beads. As such,by using constant and variable regions (i.e. “CV” or “CVC” when twoconstant regions are used) in a set of probes, an array specific set orlibrary of probes is generated.

The variable region contains a sequence that is unique to each address.Each “N” or variable position within the variable region can contain oneof ten possibilities as described above. As such, the number of uniqueaddresses in a library is determined by the length of the variableregion: if L is length of the variable region, the number of uniqueaddresses, A, is A=10^(L).

Accordingly, decoding the first base of the variable region of adecoding sequence is illustrated below:

Any address with an A at the first position would hybridize with thefourth oligonucleotide and give the corresponding color. An address withan A/C at the first position would hybridize with both the third andfourth decoders and give the corresponding combination of colors. Byserial hybridizations, each using four decoders and each interrogating adifferent position of the V region, the entire sequence of each addresscan be determined. The “N” positions in the decoders are synthesizedwith degeneracies of the four standard bases.

In one embodiment, CVC probes are incorporated into the probes of anoligonucleotide array during synthesis as is described for the additionof adapters; see U.S. Ser. No. 09/553,993, filed Apr. 20, 2000, U.S.Ser. No. 09/556,463, filed Apr. 21, 2000, U.S. Ser. No. 60/160,917,filed Oct. 22, 1999, and U.S. Ser. No. 60/135,123, filed May 20, 1999and WO 00/63437, all of which are expressly incorporated herein byreference. In a similar fashion, the CVC probes could be synthesized inthe 5-prime ends of PCR primers used to generate a PCR-product-basedarray. By incorporating different probes into the two primers of eachPCR reaction, a combinatorial expansion of addresses is possible:

The constant regions in the C₁VC₂ addresses would be different insequence from the C₃VC₄ addresses. Each would therefore be decoded withits own set of CVC complement decoders. If C₁VC₂ has a 3-base V region(1000 addresses) and C₃VC₄ has a 3-base V region (1,000 addresses), thenthe combination results in 1,000×1,000, or one million addresses.

In a preferred embodiment, there are additional ways to increase thenumber of unique or distinct tags. That is, the use of distinctattributes on each bead can be used to increase the number of codes. Inaddition, sequential decoding allows a reuse of codes in new ways. Theseattributes are independent of each other, thus allowing the number ofcodes to grow exponentially as a function of the number of decodingsteps and the number of attributes (e.g. distinct codes). However, byincreasing the amount of decoding information obtained in a singledecoding step, the number of decoding steps is markedly reduced. Byincreasing the number of attributes per decoding step, fewer decodingsteps are required for a given number of codes. Thus, in a preferredembodiment, a variety of methods are used to generate a number of codesfor use in the process of decoding the arrays, while minimizing thenecessary decoding steps. For example, a variety of different codingstrategies can be combined: thus, different “colors”, combinations ofcolors (“hues”), different intensities of colors or hues or both,different colors and different bead sizes, etc. can all be combined.Alternatively, the number of distinct codes is markedly increased.

In a preferred embodiment DBLs rely on attaching or embedding aquantitative or discrete set of physical attributes to the bead, i.e.labeling the bead. Preferred physical attributes of a bead include butare not limited to: surface “smoothness” or “roughness”, color(fluorescent and otherwise), color intensity, size, detectable chemicalmoieties, chemical reactivity, magnetization, pH sensitivity, energytransfer efficiency between dyes present, hydrophobicity,hydrophilicity, absorptivity, charge, pH sensitivity, etc. In general,physical attributes that can be measured optically are preferred,including color, size, surface smoothness etc.

In a preferred embodiment, the DBLs may be reused by having somesubpopulations of beads comprise optical signatures. In a preferredembodiment, the optical signature is generally a mixture of reporterdyes, preferably fluorescent. By varying both the composition of themixture (i.e. the ratio of one dye to another) and the concentration ofthe dye (leading to differences in signal intensity), matrices of uniqueoptical signatures may be generated. This may be done by covalentlyattaching the dyes to the surface of the beads, or alternatively, byentrapping the dye within the bead. The dyes may be chromophores orphosphors but are preferably fluorescent dyes, which due to their strongsignals provide a good signal-to-noise ratio for decoding. Suitable dyesfor use in the invention include those listed for labeling DBLs, above.

In a preferred embodiment, the encoding can be accomplished in a ratioof at least two dyes, although more encoding dimensions may be added inthe size of the beads, for example. In addition, the labels aredistinguishable from one another; thus two different labels may comprisedifferent molecules (i.e. two different fluors) or, alternatively, onelabel at two different concentrations or intensity.

In a preferred embodiment, the dyes are covalently attached to thesurface of the beads. This may be done as is generally outlined for theattachment of the bioactive agents, using functional groups on thesurface of the beads. As will be appreciated by those in the art, theseattachments are done to minimize the effect on the dye.

In a preferred embodiment, the dyes are non-covalently associated withthe beads, generally by entrapping the dyes in the pores of the beads.

Additionally, encoding in the ratios of the two or more dyes, ratherthan single dye concentrations, is preferred since it providesinsensitivity to the intensity of light used to interrogate the reporterdye's signature and detector sensitivity.

In alternative embodiments, additional encoding parameters can be added,such as microsphere size. For example, the use of different size beadsmay also allow the reuse of sets of DBLs; that is, it is possible to usemicrospheres of different sizes to expand the encoding dimensions of themicrospheres. Optical fiber arrays can be fabricated containing pixelswith different fiber diameters or cross-sections; alternatively, two ormore fiber optic bundles, each with different cross-sections of theindividual fibers, can be added together to form a larger bundle; or,fiber optic bundles with fiber of the same size cross-sections can beused, but just with different sized beads. With different diameters, thelargest wells can be filled with the largest microspheres and thenmoving onto progressively smaller microspheres in the smaller wellsuntil all size wells are then filled. In this manner, the same dye ratiocould be used to encode microspheres of different sizes therebyexpanding the number of different oligonucleotide sequences or chemicalfunctionalities present in the array. Although outlined for fiber opticsubstrates, this as well as the other methods outlined herein can beused with other substrates and with other attachment modalities as well.

A bead decoding scheme includes assigning/imbuing a single quantifiableattribute to each bead type wherein each bead type differs in thequantifiable value of that attribute. For instance, one can attach agiven number of fluorophores to a bead and quantitate the number ofattached fluorophores in the decoding process; however, in practice,attaching a “given amount” of an attribute to a bead and accuratelymeasuring the attribute may be problematic. Typically, the coefficientof variation (CV) seen in associating an attribute and the resultantmeasurement of this attribute is quite large (>20%). By coefficient ofvariation is meant the variability in labeling a bead in successivelabelings. This CV can be determined by labeling beads with a definedgiven number of label (fluorophore, for example) in multiple tests andmeasuring the resulting signal emitted by the bead. This large CV limitsthe number of useable and resolvable “levels” for any given attribute.

A more robust decoding scheme employs ratiometric rather than absolutemeasurements for segmenting a quantitative attribute into codes. Byratiometric decoding is meant labeling a bead with a ratio of labels orattributes (i.e. 1:10, 1:1, and 10:1). In theory any number of ratioscan be used so long as the difference in signals between the ratios isdetectable. This process produced smaller CVs and allowing moreattribute segmentation within a given dynamic range. Thus, in apreferred embodiment, the use of ratiometric decoding reduces thecoefficient of variability.

In addition, as will be appreciated by those in the art, ratiometricdecoding can be accomplished in a different way. In this embodiment,rather than add a given number of beads with a first dye (or dyecombination) intensity in the first decoding reaction and a secondnumber with a second dye intensity in the sequential second decodingreaction, this ratiometric analysis may be done by using a ratio oflabelled:unlabelled beads. That is, given a set saturating concentrationof decoding beads, for example 100,000 beads/reaction, the firstintensity decoding step may be done by adding 100,000 labelled beads andthe second step can be done by adding 10,000 labelled beads and 90,000unlabeled beads. Equilibrium dictates that the second step will give onetenth the signal intensity.

Because of the spread in values of a quantitatively measured attributevalue, the number of distinct codes is practically limited to less thana dozen or so codes. However, by serially “painting” (i.e. temporarilyattaching an attribute level to a bead) and “stripping” (removing theattribute level) a bead with different attribute values, the number ofpossible codes grows exponentially with the number of serial stages inthe decoding process.

An example is illustrative. For instance, 9 different bead types andthree distinguishable attribute distributions (Table 1). “Painting”(labeling) the beads with different attribute values in acombinatorially distinct pattern in the two different stages, generatesa unique code for each bead type, i.e. nine distinct codes aregenerated. Thus, in a preferred embodiment beads are labeled withdifferent attributes in a combinatorially distinct pattern in aplurality of stages. This generates unique codes for each bead type.Examples of different attributes are described above. Labeling of beadswith different attributes is performed by methods known in the art.

TABLE 1 Serial decode generates unique codes using a small number ofattribute levels. stage 1 stage 2 Bead attribute attribute Type valuevalue Code 1 L L (L, L) 2 L M (L, M) 3 L H (L, H) 4 M L (M, L) 5 M M (M,M) 6 M H (M, H) 7 H L (H, L) 8 H M (H, M) 9 H H (H, H) Number of uniquecodes = Number of attributes − Number of stages

As outlined herein, fluorescent colors are a particularly convenient andpreferable attribute to use in a decoding scheme. Fluorescent colors canbe attached to any agent that recognizes an IBL to form a labeled DBL. Afluorescently labeled oligonucleotide is a particularly useful DBL sinceit can specifically and reversibly “paint” (label) any desired subset ofbeads with a particular color simply by the process of hybridization anddehybridization (i.e. to the DBL with a complementary sequence).Moreover, fluorescence is easily imaged and quantitated using standardoptical hardware and software. In order to “paint” a given bead typewith a particular color, the bead type must be labeled with a uniquehybridizable DNA sequence (IBL) and the decoding solution must containthe color-labeled complement of that sequence.

One consideration in implementing a decoding scheme is to minimize thenumber of images collected. In a color-based scheme, the number ofimages collected is the product of the number of colors and the numberof stages. The number of images can be reduced by “painting” a bead withmultiple colors for each given stage. By assigning multiple colors to abead, the number of effective codes is increased. As an example, in a 24bit three color scheme (e.g. red, green, blue) coloring process used bycomputers, a total of 256*256*256=16.7 million different “hues” can begenerated from just three colors (red, green, blue).

Thus, in a preferred embodiment DBLs are labeled with a combination ofcolored fluorophores. As such, this method finds use in increasing thenumber of available codes for labeling DBLs using only a handful ofdifferent dyes (colors). Increasing the number of codes available ateach decoding step will greatly decrease the number of decoding stepsrequired in a given decoding process.

In one embodiment a population of oligonucleotides encoding a single DBLis labeled with a defined ratio of colors such that each bead to whichthe DBL binds is identified based on a characteristic “hue” formulatedfrom the combination of the colored fluorophores. In a preferredembodiment two distinct colors are used. In a preferred embodiment,three or more distinct dyes (colors) are available for use. In thisinstance the number of differentiable codes generated by labeling apopulation of oligonucleotides encoding a single DBL with any givencolor is three. However by allowing combinations of colors and colorlevels in the labeling, many more codes are generated.

For decoding by hybridization, a preferred number of distinguishablecolor shades is from 2 to 2000; a more preferred number ofdistinguishable color shades is from 2 to 200 and a most preferrednumber of distinguishable color shades is from 2 to 20. Utilizing threedifferent color shades (intensities) and three colors, the number ofdifferent hues will be 3⁴=81. Combining a hue with sequential decodingallows a virtually limitless number of codes to be generated.

As previously described, the DBL can be any agent that binds to the IBL.In a preferred embodiment, a single DBL is labeled with a pre-determinedratio of colors. This ratio is varied for each DBL thus allowing for aunique “hue” for each DBL labeled as such. Following treatment of thebeads with the DBL, the bead is analyzed to determine the “hue”associated with each bead, thereby identifying the bead with itsassociated bioactive agent.

For instance, with four primary colors and two intensity levels (coloris present or absent), fifteen different hues/stage are possible. Iffour dyes and three different intensity levels are used (absent,half-present, fully present), then 73 different hues/stage are possible.In this case, acquisition of only 4 color images is sufficient to obtaininformation on 73 different coding hues.

In a preferred embodiment the DBL relies on a Fluorescence ResonanceEnergy Transfer (FRET) identifier. Fluorescence resonance energytransfer (FRET) is well known in the art and has been employed forvarious biotechnological applications. FRET requires that the energy ofthe donor excited state overlap with the ground state of the acceptor,implying that the donor emission spectrum overlaps with the acceptorabsorbance spectrum. In a FRET experiment, the donor is excited withlight; some of the energy of the excited state is released as lightemission while some of the energy is transferred to the acceptor, whichbecomes excited emitting light at a characteristic wavelength. Thedistance over which energy transfer may occur can be varied by thechoice of energy donor and acceptor, wherein the use of variousdifferent donor and acceptor molecules is contemplated. The formula forthe efficiency of energy transfer is:T=R _(o) ⁶/(R _(o) ⁶ +R ⁶)

Where R is the distance between the donor and acceptor, and R₀ is aconstant related to the characteristic overlap of the absorption andemission spectra of the donor-acceptor pair.

A FRET identifier comprises a linker region that is labeled at eachterminus with a fluorophore. The first fluorophore serves as a donorfluorophore, whose signal is detectable; the other acts as an acceptorfluorophore. The emission of the acceptor fluorophore also is adetectable signal, the intensity of which varies proportionally with thedistance between the two fluorophores. That is, by varying the distancebetween the two fluorophores different codes can be generated.

Identification of the decoding oligonucleotide, therefore, isaccomplished by identifying the “efficiency” or ratio of intensities ofthe acceptor fluorophore and the donor fluorophore. This ratio should beinvariant with regard to the number of decoding oligonucleotides boundto a particle (bead); rather, without being bound by any theory, thedistance between the fluorophores determines the ratio. Thus, by varyingthe distance between the fluorophores (i.e. increasing or decreasing thelength of the linker), unique “codes” can be assigned to the DBLs. Eachcode is characterized by the ratio of emission intensities between thetwo fluorophores.

An even greater advantage is realized when different bead attributes canbe collected in a single image rather than from multiple images. Forinstance, if the colors of red, green and blue are used as attributes,three separate images need to be collected. However, if different shadesor intensities of each color are used in the coding process, a muchlarger number of attributes can be realized in a single image. Thedifficulty with using mixtures of colors, is that the color ratios canvary somewhat between bead types since individual components are mixedtogether to determine the color ratio. If on the other hand, the colorratio is built into the decoding label, as with the above described FRETdecoding oligonucleotides, the decoding process should be more reliableand more efficient.

In preferred embodiments of the present invention, the first componentmay be a lanthanide such as a chelate of europium(III) (Eu(III)) orterbium(III) (Tb(III)) which serves as a fluorescent donor and thesecond component may be an activated ester of cyanine 5 (Cy5) ortetramethylrhodamine (TMR) which serve as energy acceptors of Eu(III)and Tb(III). Other first and second component pairs that will find usefor FRET include, for example, fluorescein and rhodamine; FITC andrhodamine; and fluorescein and trinitrophenyl, and other components asare well known in the art.

The linker is comprised of any spacer element including alkyl linkers,polymer linkers, etc. For ease of synthesis, preferably the linkercomprises nucleic acids (including nucleic acid analogs).

The FRET decoding oligonucleotide comprises a spacer or linker regionthat is labeled with fluorophores (dyes). In general, this may be usedin two general ways. In a preferred embodiment, as generally depicted inFIG. 4, the linker is attached to the terminus of a decoding nucleotidethat hybridizes with (is complementary to) the IBL. In this embodiment,the linker length can be varied to adjust signal intensity. Thus, in apreferred embodiment a FRET oligonucleotide is adjoined to anoligonucleotide DBL. In one embodiment the FRET oligonucleotide issynthesized independent of the DBL oligo. Subsequently the FREToligonucleotide is ligated to the DBL oligo according to methods wellknown in the art. Alternatively, the DBL oligo and FRET oligo aresynthesized as a single oligonucleotide comprising the FRET fluorophoresat the desired positions.

Alternatively, in a second embodiment the linker is itself theoligonucleotide that hybridizes with (is complementary to) the IBL. Thatis, fluorophores are attached to the decoding oligonucleotide atdifferent basepair lengths; some may be 10 nucleotides apart, some 20,etc. In a preferred embodiment the oligonucleotide DBL is itself labeledwith fluors; as such the oligonucleotide serves as both DBL and FREToligonucleotide.

The linker region of a FRET oligonucleotide comprises any set ofnucleotides; in a preferred embodiment, for example when the linker doesnot hybridize to anything, the linker comprises a single repeatednucleotide. A single repeated nucleotide is less likely to hybridizewith the IBL or bioactive agent on the bead because of the reducedlikelihood of the presence of a complementary nucleotide sequence ineither the IBL or bioagent on the bead. In a preferred embodiment, thelinker comprises a polynucleotide linker such as poly T, poly A, poly Gor poly C, or combinations.

The linker can be of any length so long as the emission of the firstfluorophore is absorbed by the second fluorophore. In a preferredembodiment the linker is formed of from 1 to at least 100 nucleotides.More preferably the linker is formed from 1 to 70 nucleotides. Mostpreferably the linker is formed from 1 to 40 nucleotides, with from 5 to20 being especially preferred.

In a preferred embodiment, for a given DBL sequence, several codes canbe generated by varying the distance of separation of the twofluorophores and hence generating several energy transfer “efficiency”ratios.

In another preferred embodiment, for a given DBL sequence, a single codecan be generated by varying the distance of separation of the twoflourophores and hence generating a single energy transfer ratio for asingle DBL. However, numerous DBLs can be labeled with distinct FREToligos thus allowing for the simultaneous determination oridentification of multiple DBLs in a single imaging session as a resultof the plurality of energy transfer ratios.

For example, given a 40-mer decoding oligo, the donor and acceptorfluorophores can be located as close as one or as far apart as 40 bases.In principle this should allow 39 codes to be generated for a singledecoding sequence. Empirically, the number of codes generated depends ondispersion in the distributions for the ratiometric measurements.

Also, given a 40-mer decoding oligo, 39 unique codes can be generatedwhich will identify 39 different oligos.

The present invention provides array compositions comprising a substratewith a surface comprising discrete sites. A population of microspheresis distributed on the sites, and the population comprises at least afirst and a second subpopulation. Each subpopulation comprises abioactive agent, and, in addition, at least one optical dye with a givenpKa. The pkas of the different optical dyes are different.

In a preferred embodiment, when for example the array comprises clonednucleic acids, there are several methods that can be used to decode thearrays. In a preferred embodiment, when some sequence information aboutthe cloned nucleic acids is known, specific decoding probes can be madeas is generally outlined herein.

In a preferred embodiment, “random” decoding probes can be made. Bysequential hybridizations or the use of multiple labels, as is outlinedabove, a unique hybridization pattern can be generated for each sensorelement. This allows all the beads representing a given clone to beidentified as belonging to the same group. In general, this is done byusing random or partially degenerate decoding probes, that bind in asequence-dependent but not highly sequence-specific manner. The processcan be repeated a number of times, each time using a different labelingentity, to generate a different pattern of signals based onquasi-specific interactions. In this way, a unique optical signature iseventually built up for each sensor element. By applying patternrecognition or clustering algorithms to the optical signatures, thebeads can be grouped into sets that share the same signature (i.e. carrythe same probes).

In order to identify the actual sequence of the clone itself, additionalprocedures are required; for example, direct sequencing can be done. Byusing an ordered array containing the clones, such as a spotted cDNAarray, a “key” can be generated that links a hybridization pattern to aspecific clone whose position in the set is known. In this way the clonecan be recovered and further characterized.

Alternatively, clonal arrays can be decoded using binary decoding withvector tags. For example, partially randomized oligos are cloned into anucleic acid vector (e.g. plasmid, phage, etc.). Each oligonucleotidesequence consists of a subset of a limited set of sequences. Forexample, if the limites set comprises 10 sequences, each oligonucleotidemay have some subset (or all of the 10) sequences. Thus each of the 10sequences can be present or absent in the oligonucleotide. Therefore,there are 2¹⁰ or 1,024 possible combinations. The sequences may overlap,and minor variants can also be represented (e.g. A, C, T and Gsubstitutions) to increase the number of possible combinations. Anucleic acid library is cloned into a vector containing the random codesequences. Alternatively, other methods such as PCR can be used to addthe tags. In this way it is possible to use a small number of oligodecoding probes to decode an array of clones.

There are a variety of methods used to evaluate decoding. In a preferredembodiment, discriminant analysis and cluster algorithms and computerapparatus are used to analyze the decoding data from the arrays of theinvention. The potentially large number of codes utilized in theinvention, coupled with the use of different intensities and “hues” offluorophores in multi-step decoding processes, requires goodclassification of the data. The data, particularly intensity data, isacquired in a multi-step process during which beads are “painted” (forexample by hybridizing dye-labeled complementary decodingoligonucleotides to the IBL probes on the beads, or the formation ofbinding ligand pairs for non-nucleic acid IBL-DBL pairs) with differentcolors or mixtures of colors (“hues”) at each stage. The challenge is toaccurately classify a bead as to which color with which it was paintedat each step. The more closely related the “paints” (colors) are to oneanother (as determined by the optical imaging system), the moredifficult the classification.

The proximity of the dyes as seen by the imaging system is determined bythe spectral properties of the decoding dyes and the spectral channelseparation of the imaging system. Better color separation is achieved byemploying fluorescent dyes with narrow emission spectra, and byemploying an optical system with narrow band pass excitation andemission filters which are designed to excite the dye “on peak” andmeasure its emission “on peak”. The process of optically imaging thedyes on the beads is similar to the human vision process in which ourbrain sees color by measuring the ratio of excitation in the threedifferent cone types within our eye. However, with an optical imagingsystem, the number of practical color channels is much greater than thethree present in the human eye. CCD based imaging systems can “see”color from 350 nm up to 850 nm whereas the cones in the eye are tuned tothe visible spectrum from 500–600 nm.

The problem of decoding bead arrays is essentially a discriminantanalysis classification problem. Thus, in a preferred embodiment, ananalysis of variance in hyperspectral alpha space is performed on aknown set of bead colors or hues. The center of the bead clusters inalpha space are termed the centroids of the clusters, and the scatter ofthe points within a cluster determines the spread of the cluster. Arobust classification scheme requires that the distance between thecentroids of the different bead classes (hues) is much greater than thespread of any cluster class. Moreover, the location of the centroidsshould remain invariant from substrate to substrate and from experimentto experiment.

Thus, in a preferred embodiment, a hue “zone” is defined as a region inalpha space surrounding the hue centroid and extending out to the spreadradius of the cluster. Given a reference set of hue centroids and spreadradii, as determined empirically, the classification of a new set ofdata can be accomplished by asking whether a given bead point fallsclosest to or within the “zone” of a hue cluster. This is accomplishedby calculating the Mahalanobis distance (in this case, it is simply aEuclidean distance metric) of the bead point from the centroids of thedifferent hue classes. For the data shown in FIG. 6, the location of thecentroids and their distances from one another are indicated in

TABLE 2 Table 2 Distance between centroids dye/ Centroid position Bod-Bod- Bod- Bod- channel Blue Green Yellow Red 493 R6G 564 TXR Bod-4930.63 0.22 0.11 0.03 0.00 Bod-R6G 0.03 0.51 0.37 0.09 0.72 0.00 Bod-5640.06 0.04 0.57 0.32 0.81 0.55 0.00 Bod-TXR 0.09 0.05 0.04 0.82 0.99 0.930.73 0.00

For classifying the different beads into a particular hue class, aEuclidean distance cutoff of 0.3 was chosen. The closest two centroids,the Bod-R6G and Bod-564 (dist=0.55), have a slight overlap in theirdecoding zones when using a Euclidean or Mahalanobis distance of 0.3. Animprovement in classification can be achieved by decreasing thisdistance, and by weighting the different coordinate axes appropriately.

Accordingly, the present invention provides computer methods foranalyzing and classifying the color of a bead. The classification of thecolor of the bead is done by viewing the bead in hyperspectral “alpha”space (Δ₁=I₁/6I_(I), Δ₂=I₂/6I_(i), Δ₃=I₃/6I_(i), etc.) in which eachcoordinate axis represents the fraction of the bead intensity within agiven imaging channel. For instance, if four imaging channels are usedto image the beads, the color or hue of a bead can be represented by apoint in 3-D alpha space (the fourth dimension is not necessary sinceSa₁=1). Given a set of different primary dyes by which to “paint” thebeads, the number of hues that can be generated from these dyes isunlimited since the dyes can be combined in varying ratios and invarying combinatorial patterns. The number of practical hues isexperimentally determined by the separation of the different hueclusters in hyperspectral alpha space.

FIG. 6 shows a hyperspectral alpha plot of beads “painted” with fourdifferent hues imaged in four separate imaging channels. Note that thebeads form four distinct clusters. The fact that these four clusters arewell separated allows a robust decode classification scheme to beimplemented.

In a preferred embodiment, a quality control analysis of the decodingprocess is done. This is achieved by performing a cluster analysis ofalpha space for each decoding stage. The number of clusters determinedwill be fixed by the expected number of hues. The positions of thecluster centroids will be monitored and any deviations from the expectedposition will be noted.

Thus the invention provides an apparatus for decoding the arrays of theinvention. In addition to the compositions outlined herein, theapparatus includes a central processing unit which communicates with amemory and a set of input/output devices (e.g., keyboard, mouse,monitor, printer, etc.) through a bus. The general interaction between acentral processing unit, a memory, input/output devices, and a bus isknown in the art. One aspect of the present invention is directed towardthe hyperspectral “alpha” space classification system stored in thememory.

The classification system program includes a data acquisition modulethat receives data from the optical reader or confocal microscope (orother imaging system). In general, the classification program alsoincludes an analysis module, that can analyze the variance inhyperspectral alpha space, calculate the centroids of the clusters,calculate the scatter of the cluster (the spread) and define the huezone and distance cutoff. In general, the analysis module will furtherdetermine whether a data point falls within the hue zone by calculatingthe Mahalanobis distance.

Finally, the analysis module will analyze the different sequentialdecoding information to finally assign a bioactive agent to a beadlocation.

In this way, sequential decoding steps are run, with each step utilizingthe discriminant analysis calculations to assign each bead in the arrayto a hue cluster at each step. The buildup of the sequential decodinginformation allows the correlation of the location of a bead and thechemistry contained on it.

In one embodiment, the invention includes modifying the number ofdetection channels available for analysis relative to the number oflabels to be detected. That is, in one embodiment, the number ofdetection channels is decreased relative to the number of labels used.In an alternative embodiment the number of detection channels isincreased relative to the number of labels used.

When fewer detection channels are used, fewer images are required duringdecoding. As such the invention provides a method of reducing the numberof images obtained during decoding. Fluorescent dyes have broad bandabsorption and emission spectra. Accordingly, the emission of certaindyes is detected in multiple channels, generally by choosing channels tomaximize the signal. However by choosing the dyes and the channelscarefully, fewer channels that dyes may be used. As such, decoding withfewer channels than dyes, allows for detection of signals that aremaximally detected in a particular channel, and overlapping in severalchannels. Thus, for example, beads with dye-1 are read in channel-1,beads with dye-2 are read in channel-2 and beads with dye-3 show signalsin both channel-1 and channel-2. Example 7 is illustrative and theresults of the experiment are found in FIG. 11. It is noted, however,that three dyes are clearly detected and distinguished followingdetection in only two channels. Accordingly, using fewer detectionchannels than the number of dyes to be analyzed, allows for a reductionin the number of images and steps required for decoding.

In an alternative embodiment, the invention provides for usingadditional channels to increase the number of detectable codes. That is,additional readout channels are used to increase the coding/decodingcapacity of arrays by analyzing attributes of the bead in addition to anoptical signal such as fluorescence. For example, bead size, shape,density and surface characteristics can alter the light-scatteringproperties of the beads and can be detected optically. Additionalchannels that provide information independent of fluorescence areforward-scatter and side-scatter. Low-angle forward light scatterprovides some information on the relative size of beads, whereas sidelight scatter provides some information on the relative granularity ortexture of individual beads. By analogy these channels are often used incombination to distinguish the different major categories of white cellsin unseparated mammalian blood, but are useful in a wide variety ofother assays as well.

With respect to particle size, particles may simply be varied indiameter. Alternatively, particles could be assembled as doublets,triplets, or other higher-order multimers. Doublets and triplets oftenoccur spontaneously in a population, and can be enriched for byfractionation.

With respect to surface properties, in one embodiment, the inventionprovides the use of metallization. This can dramatically alter thescattering properties of a surface. One way to accomplish this is toattach gold particles (e.g. colloidal gold) to an affinity molecule,such as streptavidin or DNA, and then to bind the molecule at definedloadings to specific sites on the bead. For example, ten differentloadings could give ten new codes. Together with 100 fluorescence codes,this would allow the recognition of ˜1000 bead types in an array.Alternatively, metallization could be used for the assay readout, andfluorescence for the encoding.

With respect to magnetism, superparamagnetic beads could be deflectedaccording to the amount of magnetism, and directed to different readoutchannels—i.e. this mechanism could be used as a “pre-sorter” prior tofluorescence readout. In one version, bead subpopulations could belocalized to different regions of a reaction vessel (e.g. microtiterwell) by the use of small magnets. Each sub-population could be releasedsequentially for analysis.

Although the previously described method involving additional detectionchannels finds use in planar bead arrays, it also finds particular usein liquid arrays. By “liquid arrays” is meant a collection of beads thatare not spatially organized, but are typically encoded by fluorescencelabeling and “decoded” by readout on a flow-cytometer or equivalentinstrument. Assays are carried out in bulk in solution. Generally,liquid arrays are designed such that the assay result and decodingresult are obtained essentially simultaneously. However, because onlyoptical images are detected simultaneously, the number of codes that canbe analyzed is limited. That is, there is a limit to the number of codesthat can be used since the number of assay signals and encoding signalsmust be determined at different wavelengths at essentially the sametime. However, by using additional detection channels as describedabove, the number of codes that can be analyzed is markedly increased.

This method finds particular use in analyzing the results of genomicsbased assays such as gene expression profiling, genotyping and SNPanalysis. In such genomics based assays, it is frequreintly desirable tomake hundreds to millions of measurements simultaneously. Thus, byincreasing the number of codes that can be simultaneously detected,analysis of such assays is enhanced.

This invention provides methods of maximizing the information that isobtained from a single bead. That is, by providing ways of multiplexingassays on a single bead, multiple parameters can be simultaneouslydetected. That is, after a cycle of performing a particular assay,decoding and reading the assay results, the bead population isrecovered. The detection molecules are removed from the bead and thecycle is repeated.

In a preferred embodiment, parity analysis is used during decoding toincrease the robustness and accuracy of the system. By “parity analysis”is meant a decoding step wherein the signal of a particular element isanalyzed across a plurality of decoding stages. That is, following atleast one decoding step, but most preferably following multiple decodingsteps, the signal of an array element across the decoding stages isanalyzed. That is, the signal from a particular bead is evaluated acrossmultiple stages. Although the analysis includes any parameter that canbe obtained from the signals such as evaluating the total signalobtained across the stages, in a preferred embodiment, the parity of thesignals across the stages is analyzed.

By “parity” is meant the digital or modular readout of signals, i.e. oddor even, when binary signals are used. As depicted in FIG. 7, the digitsum of the signals across a plurality of stages is translated into aparity determination. The parity determination is then useful inevaluating the decoding process. An example is illustrative. Codes aredesigned to have an odd number of a particular signal, for example a redsignal, when viewed across all stages or decoding steps, or apre-determined plurality of stages or decoding steps. The detection ofan even number of red stages, provides an indication that an error hasoccurred at some point in decoding. When this result is obtained, thefaulty code can either be discarded, or the analysis repeated.

In a preferred embodiment the invention includes introducing a“redundant stage” into the decoding system. By “redundant stage” ismeant a stage that serves as a parity check. That is, following thedecoding stages, an additional stage is included to analyze the parity.This analysis provides an indication of the competence or validity ofthe decoding. That is, when codes are designed with a pre-determinedparity, the redundant stage is used to detect the parity of the signalsobtained from the decoding step. That is, the redundant stage detectserrors in parity because if there has been an error in decoding, theparity detected following the redundant stage will be different from theparity designed into the codes.

An example is illustrative. In the following illustration, arrayelements are decoded in two stages with two colors. This results in thedetection of 4 (2²) codes. The codes are designed to result in oddparity following the analysis with the redundant stage. That is, thecode sequences can be made such that each code will contain an oddnumber of “red” colors (when viewed across all stages, or when viewedacross the pre-determined number of stages). In the illustration below,only the stage where the “red” signal is detected is indicated.

Redundant Parity (number of Code Stage 1 Stage 2 Stage “red” stages) 1 00 red 1 (odd) 2 0 red 0 1 (odd) 3 red 0 0 1 (odd) 4 red red red 3 (odd)

However, if there is an error during decoding, this will be reflected asdetecting a parity that is different from that designed into the codes.In the following illustration the same codes as above are used however,an error is detected. In this case if one detects an even number of“red” stages it can be inferred that there has been a decoding error.

Redundant Parity (number of Code Stage 1 Stage 2 Stage “red” stages) 1 00 red 1 (odd) 2 0 red 0 1 (odd) 3 red 0 0 1 (odd) 4 red 0 red  2 (even)(error)

Although in some embodiments, the parity is binary, i.e. odd or even, inother embodiments the parity is modular or based on numbers other than2. That is, the parity can be based on detection of 3 or more signals.In a preferred embodiment, the signals are multiple colors. In aparticularly preferred embodiment the colors are red, green and blue.

An example is illustrative. In the following illustration, codes aredecoded in two steps with three colors. This results in the detection of3² (9) signals. In the illustration, red is indicated by 1, green isindicated by 2 and blue is indicated by 3. The parity is based on amodulus 3. By including a third stage as a redundant stage the paritycan be checked as described above.

Code Stage 1 Stage 2 Redundant stage Parity 1 1 1 1 3 2 1 2 3 6 3 1 3 26 4 2 1 3 6 5 2 2 2 6 6 2 3 1 6 7 3 1 2 6 8 3 2 1 6 9 3 3 3 9

However, when there is a decoding error, the parity will be incorrect;in the illustration the parity readout is not divisible by 3.

Code Stage 1 Stage 2 Redundant stage Parity 1 1 1 1 3 2 1 2 3 6 3 1 3 26 4 2 1 3 6 5 2 2 2 6 6 2 3 1 6 7 3 1 2 6 8 3 2 1 6 9 3 2 3 8 (error)

Thus, a redundant stage allows for the detection of errors on the arrayor in the signal obtained from array.

Although described above for detecting errors on arrays, the method ofusing redundant stages is not limited to arrays. That is, the methodfinds use in detecting errors in a variety of systems that send, receiveand/or interpret or analyze data such as communications systemsincluding modems etc. In particular, the method of using the modularredundant stages allows for processing of signals of vastly increasedcomplexity as compared to sending bits of data. That is, when usingmodular redundant stages, characters as opposed to bits, can be sentacross modem lines in the communications field. The modular redundantstage is used to detect errors in the communication or processing of thesignals.

In additional embodiment a redundant channel is used during thedecoding. By “redundant channel” is meant either the use of multiplefilters to analyze a particular signal or repeated use of the samefilter to analyze the signal, i.e. multiple signals from the samechannel. Redundant channels fine use as a source of a “back-up” signalof sorts for a particular array element. That is, when multiple imagesare obtained as a result of redundant channels, the likelihood ofobtaining the correct signal increases. That is, when it is realizedthat one of the results for an element is compromised for some reason,the image obtained from the redundant channel is used instead. When theinitial image in not compromised, the result from the redundant channelneed not be used, although it may be used, for example as a way toconfirm the initial signal.

An example is illustrative. During decoding, when it is apparent thatone stage fails, for example as a result of mis-registration of achannel, the use of a redundant channel serves to rescue the decoding atthat particular stage. Mis-registration can also be addressed asoutlined in U.S. Ser. No. 09/636,387, filed Aug. 9, 2000, which isexpressly incorporated herein by reference. As noted above, theredundant channel can either be an image separate filter to analyze thesignal, or alternatively, it can be multiple images taken from the samefilter.

In one embodiments, the number of redundant channels increases as thenumber of decoding stages increases. Because the investment in decodingincreases as each decoding stage or step proceeds, it is important inthis embodiment to ensure the competence of decoding at later decodingstages.

In a preferred embodiment, redundant systems are used to avoid the lossof information. It is appreciated that when “S” decoding steps(sometimes referred to herein as “stages”) and “C” colors are used, themaximum number of distinct signals is C^(S) (C raised to the “S”thpower). In some instances, the corruption of a stage results in the lossof C^(S)−(C−1)xC^((s−1)) codes. Depending on the values of C and S, theloss can be substantial. Accordingly, the present invention provides formethods of improving the robustness of the decoding system. That is, theinvention provides for a method of confirming the results of a decodinganalysis or detecting errors in a decoding analysis during or after thedecoding procedure. The method also decreases the number of falsepositive or false negative signals; similarly, the number of truepositives and/or true negatives increases.

In a preferred embodiment, several levels of redundancy are built intothe arrays of the invention. Building redundancy into an array givesseveral significant advantages, including the ability to makequantitative estimates of confidence about the data and signficantincreases in sensitivity. Thus, preferred embodiments utilize arrayredundancy. As will be appreciated by those in the art, there are atleast two types of redundancy that can be built into an array: the useof multiple identical sensor elements (termed herein “sensorredundancy”), and the use of multiple sensor elements directed to thesame target analyte, but comprising different chemical functionalities(termed herein “target redundancy”). For example, for the detection ofnucleic acids, sensor redundancy utilizes of a plurality of sensorelements such as beads comprising identical binding ligands such asprobes. Target redundancy utilizes sensor elements with different probesto the same target: one probe may span the first 25 bases of the target,a second probe may span the second 25 bases of the target, etc. Bybuilding in either or both of these types of redundancy into an array,significant benefits are obtained. For example, a variety of statisticalmathematical analyses may be done.

In addition, while this is generally described herein for bead arrays,as will be appreciated by those in the art, this techniques can be usedfor any type of arrays designed to detect target analytes.

In a preferred embodiment, sensor redundancy is used. In thisembodiment, a plurality of sensor elements, e.g. beads, comprisingidentical bioactive agents are used. That is, each subpopulationcomprises a plurality of beads comprising identical bioactive agents(e.g. binding ligands). By using a number of identical sensor elementsfor a given array, the optical signal from each sensor element can becombined and any number of statistical analyses run, as outlined below.This can be done for a variety of reasons. For example, in time varyingmeasurements, redundancy can significantly reduce the noise in thesystem. For non-time based measurements, redundancy can significantlyincrease the confidence of the data.

In a preferred embodiment, a plurality of identical sensor elements areused. As will be appreciated by those in the art, the number ofidentical sensor elements will vary with the application and use of thesensor array. In general, anywhere from 2 to thousands may be used, withfrom 2 to 100 being preferred, 2 to 50 being particularly preferred andfrom 5 to 20 being especially preferred. In general, preliminary resultsindicate that roughly 10 beads gives a sufficient advantage, althoughfor some applications, more identical sensor elements can be used.

Once obtained, the optical response signals from a plurality of sensorbeads within each bead subpopulation can be manipulated and analyzed ina wide variety of ways, including baseline adjustment, averaging,standard deviation analysis, distribution and cluster analysis,confidence interval analysis, mean testing, etc.

In a preferred embodiment, the first manipulation of the opticalresponse signals is an optional baseline adjustment. In a typicalprocedure, the standardized optical responses are adjusted to start at avalue of 0.0 by subtracting the integer 1.0 from all data points. Doingthis allows the baseline-loop data to remain at zero even when summedtogether and the random response signal noise is canceled out. When thesample is a fluid, the fluid pulse-loop temporal region, however,frequently exhibits a characteristic change in response, eitherpositive, negative or neutral, prior to the sample pulse and oftenrequires a baseline adjustment to overcome noise associated with driftin the first few data points due to charge buildup in the CCD camera. Ifno drift is present, typically the baseline from the first data pointfor each bead sensor is subtracted from all the response data for thesame bead. If drift is observed, the average baseline from the first tendata points for each bead sensor is substracted from the all theresponse data for the same bead. By applying this baseline adjustment,when multiple bead responses are added together they can be amplifiedwhile the baseline remains at zero. Since all beads respond at the sametime to the sample (e.g. the sample pulse), they all see the pulse atthe exact same time and there is no registering or adjusting needed foroverlaying their responses. In addition, other types of baselineadjustment may be done, depending on the requirements and output of thesystem used.

Once the baseline has been adjusted, a number of possible statisticalanalyses may be run to generate known statistical parameters. Analysesbased on redundancy are known and generally described in texts such asFreund and Walpole, Mathematical Statistics, Prentice Hall, Inc. NewJersey, 1980, hereby incorporated by reference in its entirety.

In a preferred embodiment, signal summing is done by simply adding theintensity values of all responses at each time point, generating a newtemporal response comprised of the sum of all bead responses. Thesevalues can be baseline-adjusted or raw. As for all the analysesdescribed herein, signal summing can be performed in real time or duringpost-data acquisition data reduction and analysis. In one embodiment,signal summing is performed with a commercial spreadsheet program(Excel, Microsoft, Redmond, Wash.) after optical response data iscollected.

In a preferred embodiment, cummulative response data is generated bysimply adding all data points in successive time intervals. This finalcolumn, comprised of the sum of all data points at a particular timeinterval, may then be compared or plotted with the individual beadresponses to determine the extent of signal enhancement or improvedsignal-to-noise ratios.

In a preferred embodiment, the mean of the subpopulation (i.e. theplurality of identical beads) is determined, using the well knownEquation 1:

$\begin{matrix}{\mu = {\sum\;\frac{x_{i}}{n}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

In some embodiments, the subpopulation may be redefined to exclude somebeads if necessary (for example for obvious outliers, as discussedbelow).

In a preferred embodiment, the standard deviation of the subpopulationcan be determined, generally using Equation 2 (for the entiresubpopulation) and Equation 3 (for less than the entire subpopulation):

$\begin{matrix}{\sigma = \sqrt{\frac{\sum\left( {x_{i} - \mu} \right)^{2}}{n}}} & {{Equation}\mspace{14mu} 2} \\{s = \sqrt{\frac{\sum\left( {x_{i} - \overset{\_}{x}} \right)^{2}}{n - 1}}} & {{Equation}\mspace{14mu} 3}\end{matrix}$

As for the mean, the subpopulation may be redefined to exclude somebeads if necessary (for example for obvious outliers, as discussedbelow).

In a preferred embodiment, statistical analyses are done to evaluatewhether a particular data point has statistical validity within asubpopulation by using techniques including, but not limited to, tdistribution and cluster analysis. This may be done to statisticallydiscard outliers that may otherwise skew the result and increase thesignal-to-noise ratio of any particular experiment. This may be doneusing Equation 4:

$\begin{matrix}{t = \frac{\overset{\_}{x} - \mu}{s/\sqrt{n}}} & {{Equation}\mspace{14mu} 4}\end{matrix}$

In a preferred embodiment, the quality of the data is evaluated usingconfidence intervals, as is known in the art. Confidence intervals canbe used to facilitate more comprehensive data processing to measure thestatistical validity of a result.

In a preferred embodiment, statistical parameters of a subpopulation ofbeads are used to do hypothesis testing. One application is testsconcerning means, also called mean testing. In this application,statistical evaluation is done to determine whether two subpopulationsare different. For example, one sample could be compared with anothersample for each subpopulation within an array to determine if thevariation is statistically significant.

In addition, mean testing can also be used to differentiate twodifferent assays that share the same code. If the two assays giveresults that are statistically distinct from each other, then thesubpopulations that share a common code can be distinguished from eachother on the basis of the assay and the mean test, shown below inEquation 5:

$\begin{matrix}{z = \frac{{\overset{\_}{x}}_{1} - {\overset{\_}{x}}_{2}}{\sqrt{\frac{\sigma_{1}^{2}}{n_{1}} + \frac{\sigma_{2}^{2}}{n_{2}}}}} & {{Equation}\mspace{14mu} 5}\end{matrix}$

Furthermore, analyzing the distribution of individual members of asubpopulation of sensor elements may be done. For example, asubpopulation distribution can be evaluated to determine whether thedistribution is binomial, Poisson, hypergeometric, etc.

In addition to the sensor redundancy, a preferred embodiment utilizes aplurality of sensor elements that are directed to a single targetanalyte but yet are not identical. For example, a single target nucleicacid analyte may have two or more sensor elements each comprising adifferent probe. This adds a level of confidence as non-specific bindinginteractions can be statistically minimized. When nucleic acid targetanalytes are to be evaluated, the redundant nucleic acid probes may beoverlapping, adjacent, or spatially separated. However, it is preferredthat two probes do not compete for a single binding site, so adjacent orseparated probes are preferred. Similarly, when proteinaceous targetanalytes are to be evaluated, preferred embodiments utilize bioactiveagent binding agents that bind to different parts of the target. Forexample, when antibodies (or antibody fragments) are used as bioactiveagents for the binding of target proteins, preferred embodiments utilizeantibodies to different epitopes.

In this embodiment, a plurality of different sensor elements may beused, with from about 2 to about 20 being preferred, and from about 2 toabout 10 being especially preferred, and from 2 to about 5 beingparticularly preferred, including 2, 3, 4 or 5. However, as above, moremay also be used, depending on the application.

As above, any number of statistical analyses may be run on the data fromtarget redundant sensors.

One benefit of the sensor element summing (referred to herein as “beadsumming” when beads are used), is the increase in sensitivity that canoccur.

Once made, the compositions of the invention find use in a number ofapplications. In a preferred embodiment, the compositions are used toprobe a sample solution for the presence or absence of a target analyte,including the quantification of the amount of target analyte present. By“target analyte” or “analyte” or grammatical equivalents herein is meantany atom, molecule, ion, molecular ion, compound or particle to beeither detected or evaluated for binding partners. As will beappreciated by those in the art, a large number of analytes may be usedin the present invention; basically, any target analyte can be usedwhich binds a bioactive agent or for which a binding partner (i.e. drugcandidate) is sought.

Suitable analytes include organic and inorganic molecules, includingbiomolecules. When detection of a target analyte is done, suitabletarget analytes include, but are not limited to, an environmentalpollutant (including pesticides, insecticides, toxins, etc.); a chemical(including solvents, polymers, organic materials, etc.); therapeuticmolecules (including therapeutic and abused drugs, antibiotics, etc.);biomolecules (including hormones, cytokines, proteins, nucleic acids,lipids, carbohydrates, cellular membrane antigens and receptors (neural,hormonal, nutrient, and cell surface receptors) or their ligands, etc);whole cells (including procaryotic (such as pathogenic bacteria) andeukaryotic cells, including mammalian tumor cells); viruses (includingretroviruses, herpesviruses, adenoviruses, lentiviruses, etc.); andspores; etc. Particularly preferred analytes are nucleic acids andproteins.

In a preferred embodiment, the target analyte is a protein. As will beappreciated by those in the art, there are a large number of possibleproteinaceous target analytes that may be detected or evaluated forbinding partners using the present invention. Suitable protein targetanalytes include, but are not limited to, (1) immunoglobulins; (2)enzymes (and other proteins); (3) hormones and cytokines (many of whichserve as ligands for cellular receptors); and (4) other proteins.

In a preferred embodiment, the target analyte is a nucleic acid. Theseassays find use in a wide variety of applications. Such applications aredescribed in more detail in U.S. Ser. No. 60/130,089, filed Apr. 20,1999; U.S. Ser. No. 60/160,927, filed Oct. 22, 1999, U.S. Ser. No.09/513,362, filed Feb. 25, 2000, U.S. Ser. No. 60/135,051 filed May 20,1999, U.S. Ser. No. 60/161,148, filed Oct. 22, 1999, U.S Ser. No.09/517,945, filed Mar. 3, 2000, U.S. Ser. No. 09/425,633, filed Oct. 22,1999, U.S. Ser. No. 09/535,854, filed Mar. 27, 2000, U.S. Ser. No09/535,993, filed Apr. 20, 2000, U.S. Ser. No. 09/556,463, filed Apr.21, 2000, U.S. Ser. No. 60/224,119, filed Oct. 26, 2000, all of whichare expressly incorporated herein by reference.

In a preferred embodiment, the probes are used in genetic diagnosis. Forexample, probes can be made using the techniques disclosed herein todetect target sequences such as the gene for nonpolyposis colon cancer,the BRCA1 breast cancer gene, P53, which is a gene associated with avariety of cancers, the Apo E4 gene that indicates a greater risk ofAlzheimer's disease, allowing for easy presymptomatic screening ofpatients, mutations in the cystic fibrosis gene, cytochrome p450s or anyof the others well known in the art.

In an additional embodiment, viral and bacterial detection is done usingthe complexes of the invention. In this embodiment, probes are designedto detect target sequences from a variety of bacteria and viruses. Forexample, current blood-screening techniques rely on the detection ofanti-HIV antibodies. The methods disclosed herein allow for directscreening of clinical samples to detect HIV nucleic acid sequences,particularly highly conserved HIV sequences. In addition, this allowsdirect monitoring of circulating virus within a patient as an improvedmethod of assessing the efficacy of anti-viral therapies. Similarly,viruses associated with leukemia, HTLV-I and HTLV-II, may be detected inthis way. Bacterial infections such as tuberculosis, chlamydia and othersexually transmitted diseases, may also be detected.

In a preferred embodiment, the nucleic acids of the invention find useas probes for toxic bacteria in the screening of water and food samples.For example, samples may be treated to lyse the bacteria to release itsnucleic acid, and then probes designed to recognize bacterial strains,including, but not limited to, such pathogenic strains as, Salmonella,Campylobacter, Vibrio cholerae, Leishmania, enterotoxic strains of E.coli, and Legionnaire's disease bacteria. Similarly, bioremediationstrategies may be evaluated using the compositions of the invention.

In a further embodiment, the probes are used for forensic “DNAfingerprinting” to match crime-scene DNA against samples taken fromvictims and suspects.

In an additional embodiment, the probes in an array are used forsequencing by hybridization.

The present invention also finds use as a methodology for the detectionof mutations or mismatches in target nucleic acid sequences, as isdescribed in more detail in U.S. Ser. No. 09/425,633, filed Oct. 22,1999, U.S. Ser. No. 09/535,854, filed Mar. 27, 2000, both of which areexpressly incorporated herein by reference. For example, recent focushas been on the analysis of the relationship between genetic variationand phenotype by making use of polymorphic DNA markers. Previous workutilized short tandem repeats (STRs) as polymorphic positional markers;however, recent focus is on the use of single nucleotide polymorphisms(SNPs). Common SNPs occur at an average frequency of more than 1 perkilobase in human genomic DNA. Some SNPs, particularly those in andaround coding sequences, are likely to be the direct cause oftherapeutically relevant phenotypic variants. There are a number of wellknown polymorphisms that cause clinically important phenotypes; forexample, the apoE2/3/4 variants are associated with different relativerisk of Alzheimer's and other diseases (see Cordor et al., Science261(1993). Multiplex PCR amplification of SNP loci with subsequenthybridization to oligonucleotide arrays has been shown to be an accurateand reliable method of simultaneously genotyping at least hundreds ofSNPs; see Wang et al., Science, 280:1077 (1998); see also Schafer etal., Nature Biotechnology 16:33–39 (1998). The compositions of thepresent invention may easily be substituted for the arrays of the priorart.

In a preferred embodiment, the compositions of the invention are used toscreen bioactive agents to find an agent that will bind, and preferablymodify the function of, a target molecule. As above, a wide variety ofdifferent assay formats may be run, as will be appreciated by those inthe art. Generally, the target analyte for which a binding partner isdesired is labeled; binding of the target analyte by the bioactive agentresults in the recruitment of the label to the bead, with subsequentdetection.

In a preferred embodiment, the binding of the bioactive agent and thetarget analyte is specific; that is, the bioactive agent specificallybinds to the target analyte. By “specifically bind” herein is meant thatthe agent binds the analyte, with specificity sufficient todifferentiate between the analyte and other components or contaminantsof the test sample. However, as will be appreciated by those in the art,it will be possible to detect analytes using binding which is not highlyspecific; for example, the systems may use different binding ligands,for example an array of different ligands, and detection of anyparticular analyte is via its “signature” of binding to a panel ofbinding ligands, similar to the manner in which “electronic noses” work.This finds particular utility in the detection of chemical analytes. Thebinding should be sufficient to remain bound under the conditions of theassay, including wash steps to remove non-specific binding, although insome embodiments, wash steps are not desired; i.e. for detecting lowaffinity binding partners. In some embodiments, for example in thedetection of certain biomolecules, the dissociation constants of theanalyte to the binding ligand will be less than about 10⁻⁴–10⁻⁶ M⁻¹,with less than about 10⁻⁵ to 10⁻⁹ M⁻¹ being preferred and less thanabout 10⁻⁷–10⁻⁹ M⁻¹ being particularly preferred.

Generally, a sample containing a target analyte (whether for detectionof the target analyte or screening for binding partners of the targetanalyte) is added to the array, under conditions suitable for binding ofthe target analyte to at least one of the bioactive agents, i.e.generally physiological conditions. The presence or absence of thetarget analyte is then detected. As will be appreciated by those in theart, this may be done in a variety of ways, generally through the use ofa change in an optical signal. This change can occur via many differentmechanisms. A few examples include the binding of a dye-tagged analyteto the bead, the production of a dye species on or near the beads, thedestruction of an existing dye species, a change in the opticalsignature upon analyte interaction with dye on bead, or any otheroptically interrogatable event.

In a preferred embodiment, the change in optical signal occurs as aresult of the binding of a target analyte that is labeled, eitherdirectly or indirectly, with a detectable label, preferably an opticallabel such as a fluorochrome. Thus, for example, when a proteinaceoustarget analyte is used, it may be either directly labeled with a fluor,or indirectly, for example through the use of a labeled antibody.Similarly, nucleic acids are easily labeled with fluorochromes, forexample during PCR amplification as is known in the art. Alternatively,upon binding of the target sequences, a hybridization indicator may beused as the label. Hybridization indicators preferentially associatewith double stranded nucleic acid, usually reversibly. Hybridizationindicators include intercalators and minor and/or major groove bindingmoieties. In a preferred embodiment, intercalators may be used; sinceintercalation generally only occurs in the presence of double strandednucleic acid, only in the presence of target hybridization will thelabel light up. Thus, upon binding of the target analyte to a bioactiveagent, there is a new optical signal generated at that site, which thenmay be detected.

Alternatively, in some cases, as discussed above, the target analytesuch as an enzyme generates a species that is either directly orindirectly optically detectable.

Furthermore, in some embodiments, a change in the optical signature maybe the basis of the optical signal. For example, the interaction of somechemical target analytes with some fluorescent dyes on the beads mayalter the optical signature, thus generating a different optical signal.

As will be appreciated by those in the art, in some embodiments, thepresence or absence of the target analyte may be done using changes inother optical or non-optical signals, including, but not limited to,surface enhanced Raman spectroscopy, surface plasmon resonance,radioactivity, etc.

The assays may be run under a variety of experimental conditions, aswill be appreciated by those in the art. A variety of other reagents maybe included in the screening assays. These include reagents like salts,neutral proteins, e.g. albumin, detergents, etc which may be used tofacilitate optimal protein-protein binding and/or reduce non-specific orbackground interactions. Also reagents that otherwise improve theefficiency of the assay, such as protease inhibitors, nucleaseinhibitors, anti-microbial agents, etc., may be used. The mixture ofcomponents may be added in any order that provides for the requisitebinding. Various blocking and washing steps may be utilized as is knownin the art.

In a preferred embodiment, two-color competitive hybridization assaysare run. These assays can be based on traditional sandwich assays. Thebeads contain a capture sequence located on one side (upstream ordownstream) of the SNP, to capture the target sequence. Two SNPallele-specific probes, each labeled with a different fluorophor, arehybridized to the target sequence. The genotype can be obtained from aratio of the two signals, with the correct sequence generally exhibitingbetter binding. This has an advantage in that the target sequence itselfneed not be labeled. In addition, since the probes are competing, thismeans that the conditions for binding need not be optimized. Underconditions where a mismatched probe would be stably bound, a matchedprobe can still displace it. Therefore the competitive assay can providebetter discrimination under those conditions. Because many assays arecarried out in parallel, conditions cannot be optimzed for every probesimultaneously. Therefore, a competitive assay system can be used tohelp compensate for non-optimal conditions for mismatch discrimination.

In a preferred embodiment, dideoxynucleotide chain-terminationsequencing is done using the compositions of the invention. In thisembodiment, a DNA polymerase is used to extend a primer usingfluorescently labeled ddNTPs. The 3′ end of the primer is locatedadjacent to the SNP site. In this way, the single base extension iscomplementary to the sequence at the SNP site. By using four differentfluorophors, one for each base, the sequence of the SNP can be deducedby comparing the four base-specific signals. This may be done in severalways. In a first embodiment, the capture probe can be extended; in thisapproach, the probe must either be synthesized 5′-3′ on the bead, orattached at the 5′ end, to provide a free 3′ end for polymeraseextension. Alternatively, a sandwich type assay can be used; in thisembodiment, the target is captured on the bead by a probe, then a primeris annealed and extended. Again, in the latter case, the target sequenceneed not be labeled. In addition, since sandwich assays require twospecific interactions, this provides increased specificity which isparticularly helpful for the analysis of complex samples.

In addition, when the target analyte and the DBL both bind to the agent,it is also possible to do detection of non-labelled target analytes viacompetition of decoding.

In a preferred embodiment, the methods of the invention are useful inarray quality control. Prior to this invention, no methods have beendescribed that provide a positive test of the performance of every probeon every array. Decoding of the array not only provides this test, italso does so by making use of the data generated during the decodingprocess itself. Therefore, no additional experimental work is required.The invention requires only a set of data analysis algorithms that canbe encoded in software.

The quality control procedure can identify a wide variety of systematicand random problems in an array. For example, random specks of dust orother contaminants might cause some sensors to give an incorrectsignal-this can be detected during decoding. The omission of one or moreagents from multiple arrays can also be detected; that is, since thearrays formulated herein are randomly self-assembled, arrays comprisingdifferent statistical distributions of each sensor elements may be made;the invention allows actual QC on the arrays, for “sorting” arrays withgood distributions from those with less than ideal distributions. Theseprocedures also allow the determination of sites that do not comprisebeads. This quality control procedure can also be used in non-randomarrays.

An advantage of this quality control procedure is that it can beimplemented immediately prior to the assay itself (or after, in someinstances), and is a true functional test of each individual sensor.Therefore any problems that might occur between array assembly andactual use can be detected. In applications where a very high level ofconfidence is required, and/or there is a significant chance of sensorfailure during the experimental procedure, decoding and quality controlcan be conducted both before and after the actual sample analysis.

In an alternative method of quality control, a single fiber bundle of anarray is dedicated to quality control. That is a dedicated bundle orsubset of fibers within an array is independently analyzed for qualitycontrol. In one embodiment, the control fiber(s) are targeted or labeledwith a unique code or label. At each state of hybridization, in additionto the experimental probes, the quality control probes are hybridizedwith the array. Upon taking the image from the first hybridization ofthe first stage, the intensity of the control fiber(s) is analyzed. Anaberrant reading or signal from the control fiber indicates a failed orat least non-optimal hybridization step.

In a preferred embodiment, the arrays can be used to do reagent qualitycontrol. In many instances, biological macromolecules are used asreagents and must be quality controlled. For example, large sets ofoligonucleotide probes may be provided as reagents. It is typicallydifficult to perform quality control on large numbers of differentbiological macromolecules. For example, when large populations ofdifferent random oligonucleotides are synthesized, it may be desirableto check that each population is represented. The approach describedhere can be used to do this by treating the reagents (formulated as theDBLs) as variable instead of the arrays.

In a preferred embodiment, the methods outlined herein are used in arraycalibration. For many applications, such as mRNA quantitation, it isdesirable to have a signal that is a linear response to theconcentration of the target analyte, or, alternatively, if non-linear,to determine a relationship between concentration and signal, so thatthe concentration of the target analyte can be estimated. For example,different probes can show differences in hybridization efficiencies,etc., and it may be desirable to choose appropriate probes underexperimental conditions. Thus, for example, when expression profiling isdone, and quantitation is desirable, calibration curves can be done tosee how individual probes react, and then the probes that give the bestresponse (i.e. linearity at the concentrations and conditions ofinterest) can be chosen for further assays. Accordingly, the presentinvention provides methods of creating calibration curves in parallelfor multiple beads in an array. The calibration curves can be createdunder conditions that simulate the complexity of the sample to beanalyzed. Each curve can be constructed independently of the others(e.g. for a different range of concentrations), but at the same time asall the other curves for the array.

Thus, in these embodiments, different types of experiments can be done.For example, the sequential decoding scheme can be implemented withdifferent concentrations being used as the code “labels”, rather thandifferent fluorophores. In this way, signal as a response toconcentration can be measured for each bead. This calibration can becarried out just prior to array use, so that every probe on every arrayis individually calibrated as needed. Alternatively, differentconcentrations of the decoding probe can have different labels.

It should be noted that the assay calibration methods find use innon-random arrays, as well; that is, other types of support-boundnucleic acid arrays can be calibrated using these methods as well. Thus,for example, the sequential addition of different pools of probes,wherein the concentration of the probes is varied, to biochips can allowcalibration of any assay system. This type of analysis can also be doneon non-random arrays for quality control, to verify the integrity andsequence of the support bound probes, and in assay development toidentify good probes.

In a preferred embodiment, the methods of the invention can be used inassay development as well. Thus, for example, the methods allow theidentification of good and bad probes; as is understood by those in theart, some probes do not function well because they do not hybridizewell, or because they cross-hybridize with more than one sequence. Theseproblems are easily detected during decoding. The ability to rapidlyassess probe performance has the potential to greatly reduce the timeand expense of assay development. Thus, probes that respond linearlywith concentration, show low non-specific binding, or give signals in aparticular range, can be chosen for addition to a new array for assays.

Similarly, in a preferred embodiment, the methods of the invention areuseful in quantitation in assay development. Major challenges of manyassays is the ability to detect differences in analyte concentrationsbetween samples, the ability to quantitate these differences, and tomeasure absolute concentrations of analytes, all in the presence of acomplex mixture of related analytes. An example of this problem is thequantitation of a specific mRNA in the presence of total cellular mRNA.One approach that has been developed as a basis of mRNA quantitationmakes use of a multiple match and mismatch probe pairs (Lockhart et al.,1996), hereby incorporated by reference in its entirety. While thisapproach is simple, it requires relatively large numbers of probes. Inthis approach, a quantitative response to concentration is obtained byaveraging the signals from a set of different probes to the gene orsequence of interest. This is necessary because only some probes respondquantitatively, and it is not possible to predict these probes withcertainty. In the absence of prior knowledge, only the average responseof an appropriately chosen collection of probes is quantitative.However, in the present invention, that can be applied generally tonucleic acid based assays as well as other assays. In essence, theapproach is to identify the probes that respond quantitatively in aparticular assay, rather than average them with other probes. This isdone using the array calibration scheme outlined above, in whichconcentration-based codes are used. Advantages of this approach include:fewer probes are needed; the accuracy of the measurement is lessdependent on the number of probes used; and that the response of thesensors is known with a high level of certainty, since each and everysequence can be tested in an efficient manner. It is important to notethat probes that perform well are selected empirically, which avoids thedifficulties and uncertainties of predicting probe performance,particularly in complex sequence mixtures. In contrast, in experimentsdescribed to date with ordered arrays, relatively small numbers ofsequences are checked by performing quantitative spiking experiments, inwhich a known mRNA is added to a mixture.

In a preferred embodiment, cDNA arrays are made for RNA expressionprofiling. In this embodiment, individual cDNA clones are amplified (forexample, using PCR) from cDNA libraries propagated in a host-vectorsystem. Each amplified DNA is attached to a population of beads.Different populations are mixed together, to create a collection ofbeads representing the cDNA library. The beads are arrayed, decoded asoutlined above, and used in an assay (although as outlined herein,decoding may occur after assay as well). The assay is done using RNAsamples (whole cell or mRNA) that are extracted, labeled if necessary,and hybridized to the array. Comparative analysis allows the detectionof differences in the expression levels of individual RNAs. Comparisonto an appropriate set of calibration standards allows quantification ofabsolute amounts of RNA.

The cDNA array can also be used for mapping, e.g. to mapdeletions/insertions or copy number changes in the genome, for examplefrom tumors or other tissue samples. This can be done by hybridizinggenomic DNA. Instead of cDNAs (or ESTs, etc.), other STS (sequencetagged sites), including random genomic fragments, can also be arrayedfor this purpose.

In one embodiment, the invention provides a method of single nucleotidepolymorphism (SNP) genotyping. As described herein and in U.S. Ser. No.09/553,993 filed Apr. 20, 2000, U.S. Ser. No. 09/556,463 filed Apr. 21,2000 and U.S. Ser. No. 60/244,119, filed Oct. 26, 2000, all of which areexpressly incorporated herein by reference, a target nucleic acid iscontacted with a probe that is specific for a particular allele of asingle nucleotide polymorphism. The target nucleic acid can beimmobilized prior to contacting with the probe, or alternatively, theprobe: target hybridization can occur in solution following byimmobilization of the probe: target nucleic acid complex. Alternatively,a capture prove on a bead may be specific for a particular SNP. What isimportant in this invention is the nature of the probe for detecting aSNP.

There are four possibilities for the bases at a polymorphic site: -A, T,G, or C. However, it is known that most of the human SNP's aredimorphic, i.e., they only include the combinations of two of the fourbases. As such, the prior art generally relied on distinguishing betweentwo alleles that are present within a SNP. and characterized them aseither “M”, “AB, or “BB”.

However, according to the present invention, the probes are designedsuch that each of the four possible nucleotides that could be present ata particular position are included in the SNP probes. In addition, theprobes are labeled with a specific color that corresponds with thenucleotide that hybridizes to the nucleotide to be interrogated. Forinstance, all the alleles that correspond to an A-polymorphic site arelabeled with red. Similarly, all the alleles that correspond to aG-polymorphic site are labeled green, etc. As such the true identity ofthe polymorphism is determined. That is, if the polymorphism is of thetype GT, one would not classify is as “AB” genotype. Instead, the GTcode will come up directly from the genotyping routine.

An advantage of this method over the prior art is the capability ofindependent error checking. That is, knowing that polymorphisms aretypically dimorphic, by using probes that correspond to allpossibilities of polymorphisms at a sites, one obtains a check againstfalse results, for example of a signal is detected that does notcorrespond with the known polymorphisms. That is, in the prior art, theresultant genotyping would be one of the following three possibilities:AA, AB, or BB. However, by using this invention, the number of possibleresults is increased from 3 to 10: AA, AG, AC, AT, GG, GC, GT, CC, CT,TT. Of these ten possibilities, only 3 are the possible combinations foreach SNP, e.g., a T/G polymorphism can only render one of the followingthree possibilities: TT, TG, or GG. Therefore, in this case, if thecomputed genotype falls into any of the remaining 10−3=7 combinations(e.g., either of AA, AG, AC, AT, GC, CC, CT), it would be a clearindication that an error has occurred.

All references cited herein are incorporated by reference in theirentirety.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference in theirentirety.

EXAMPLES Example 1

Sixteen microspheres (beads) were labeled combinatorially with twodifferent fluorophores (FAM and Cy3). In a first round of labeling,either FAM or Cy3 labeled oligonucleotides that were complementary tothe oligonucleotide (IBL) on the microsphere, were hybridized with themicrosphere. Labeling of oligonucleotides was performed as is well knownin the art. Hybridization conditions are known in the art.

Following a first round of hybridization, the two pools of beads weredivided into two pools each and each labeled either with the FAM or Cy3labeled oligonucleotide. This process was repeated two additional times.Thus, following four successive rounds of labeling, each microsphere waslabeled with a unique code (see FIG. 1). The identity of eachmicrosphere was elucidated by determining the identity of eachfluorophore in succession; the terminal fluorophere was determined andthen removed to allow for the identification of the next fluorophore. Inthis fashion, with as few as 4 decoding steps, the identity of 16microspheres is determined.

Example 2

A decoding scheme similar to that described in Example 1 was implementedfor four color decoding. In this example, beads were labeled asdescribed in Example 1 with the exception that 4 labels were used ateach stage. 4013 beads were labeled using Bod493, BodR6G, Bod564 andBodTXR labeled oligonucleotides. 128 different bead types wereidentified based on the successive decoding of the four colors.

Example 3

An alternative method to using multiple colors is to use ratiometricintensities as a coding scheme. A normalizing image is acquired in whichevery bead exhibits its “full” intensity. Subsequent decode stagesgenerate intensity codes by hybridizing mixtures of“labeled”:“unlabeled” complementary oligonucleotides. For instance, FIG.1 depicts three different intensity shades (low, medium, and high) whichcan be ratioed to a stage with all complements present at a “high”shading value. An experiment using grey scale decoding on 16 differentbead types is shown in FIG. 3.

FIG. 3A depicts the combinatorial pooling scheme for labeling beads withdifferent ratios of labeled oligonucleotides. A particular oligo ispresent at either 100% Cy3-labeled, 40% Cy3-labeled (60% unlabeled), or10% Cy3-labeled (90% unlabeled) fraction. Decode oligos were hybridizedto the array for 2 min. at a 50 nM concentration. Subsequently, twoindependent normalizing images (all oligo complements are present as100% Cy3-labeled species) were acquired, and the resulting beadintensities compared. This is depicted in FIG. 3B as the normalizedvalues are plotted against each other. Finally, to identify or decodethe beads, the alpha values (ratio of bead intensity in indicated decodestage to intensity in normalization image) are plotted for three decodestages described in (A). In stage 1, only two peaks are observed in thealpha value histogram since only 16 bead types are present on the array.Three distinguishable peaks are observed in the second and third decodestages indicating the feasibility of grey scale decoding.

Physical attributes and different “levels” of the attributes can be usedas codes by which to distinguish bead types from another. Thus, for anattribute to act as a robust code, it should be possible to imbue a beadwith different “levels” of a particular attribute. Each “level” of anattribute should be quantitatively well separated from other “levels”.The important point is to maximize the dynamic range of the attributemeasurement, and minimize the spread of the measurement.

Example 4

For a given oligo sequence, several codes can be generated by varyingthe distance of separation of the two fluorophores. As shown in FIG. 4,a complementary sequence containing FRET dyes separated by a linker ofvarying lengths allows multiple codes to be generated (FIG. 4A). As anexample, Probe A and B are hybridized to a complementary sequenceimmobilized on a solid support. Probe A contains dyes separated by an 8poly T linker, whereas probe B is hybridized to a linker with a 4 poly Tlinker. The efficiency of energy transfer (ratio of intensities ofdye2:dye1) is greater for probe B than for probe A (FIG. 4B).

Example 5

The following FRET oligonucleotides (probes ET1, ET2, ET5 and ET7) weresynthesized as is known in the art and labeled with Cy3 and fluorsceinseparated by linkers of varying length:

ET1 T**G*CACGAGAATGGAGGTATCT ET2 C**TGTCGC*ACGAGAATGGAGGTATCT ET5C**TGTCGGGGCACTCATTTGTGC*ACGAGAATGGAGGTATCT ET7C**TGTCGGGGCACTCATTTGTCTGTCGGGGCGC*ACGAGAATGGA GGTATCT. **is Cy3 *isfluorescein

An aliquot of each FRET oligonucleotide was assayed as is known in theart. Emission spectra were normalized relative to emission at 530 nm. Ascan be seen in FIG. 5 emission of fluorescein increases as the linker isshortened. The ratio of intensities of the two fluorophores, therefore,is characteristic of the distance between the fluorophores.

Example 6

To illustrate the primer extension decoding, a two-color model isillustrated in FIG. 9. To decode 16 different bead addresses using onlytwo colors. 16 different beads are labeled by attaching 16 differentunique oligos (IBLs) to each of 16 batches of beads and pooling the beadbatches together. Along with the beads 16 oligos (DBLs) that arecomplementary to these IBLs are synthesized. Each unique IBL used inthis method contains two domains. The domains are composed of a bindingdomain and an extension domain. The pooled bead populations are thenself-assembled onto a fiber optic tip end as described herein. Afterarray self-assembly a combinatorial color decoding process is initiatedas follows: A primer extension reaction is initiated at the end of thefiber bundle tip containing the arrayed beads by simultaneouslyhybridizing 16 complementary oligos (DBLs) to their cognate IBL. In thefirst extension stage, the DBLs would have exactly the same length (n)as the binding domain of the IBL. The first stage primer extensionreaction would contain 16 primer oligos, a DNA polymerase, appropriatebuffer, and two fluorescently-labeled dideoxynucleotide terminators.During the extension reaction, hybridized primers are extended by asingle nucleotide complementary to the first nucleotide in the extensiondomain of the immobilized IBL. The described extension reaction ‘paints’or labels any given bead with one of two possible fluorescent terminatorcolors. After the reaction is over the array tip is imaged to capturethe color of the beads at this stage. Beads are then immediatelystripped of color or label by denaturing and washing off the firstextended primer at each zip code loci. This process can be repeatedthrough multiple stages. In subsequent stages, the primer extensionreactions contain the same reagents as the previous extension reactionexcept that the binding complementary oligos are one base longer. These(n+1) primers hybridize with their cognate binding domain and one baseinto the ‘extension domain’. FIG. 9 illustrates a four-stage extensionprocess (2-color) whereby each stage allows for the assignment of aunique combinatorial color-coded ‘address’ to each bead type.

In addition, if this primer-based extension method were to be combinedwith degenerate DBL-BL combinations for decoding large combinatoriallibraries, for example using the CVC (constant-variable-constant domainidea), as described herein, the extension reaction would extend into oneof 10 different possible nucleotide combinations in a ‘variableextension region’. This would require only three extension reactions todecodify 1000 unique addresses (i.e. A=lot where L is the length of thevariable region). The combination of the degenerate DBL-IBL combinationssuch as CVC with the primer extension method is illustrated in FIG. 10.

Example 7

This method relates to fluorescent based encoding and decoding processbased on the fact that fluorescent dyes have broad band absorption andemission spectra. Fewer detection channels are used than the number ofencoding dyes used in the process. That is, for n detection channels, aparticular dye, x, can be represented by a vector I_(x) in then-dimensional space.

$I_{x}\left\{ {\frac{l}{\underset{i\mspace{14mu} I}{\overset{n}{f\mspace{14mu} I_{i}}}}\bot{I_{1}I_{2}I_{3}\ldots\; I_{n}}} \right.$where I_(i) is the intensity detected at channel i. Theoretically, aninfinite number of vectors are possible in the n-dimensional space whenn is larger or equal to 2. With an appropriate dye selection and opticalfilter design, for any given number of detection channels, a largernumber of dyes can be used for decoding. For example, to distinguishthree species, it is possible to tag the species with three differentdyes, x, y, z and use only two detection channels, such thatI _(x)={1 0}I _(y) ={a 1−a} 0<a<1I _(z)={0 1}

FIG. 11 illustrates the experimental results for this example schemeused in decoding. Three classes of beads were tagged with Bodipy-493,Bodipy-R6G and Bodipy564. Detection channel 1 consisted of a 410 nm/20excitation filter and a 540 nm/20 emission filter. Detection channel 2consisted of a 535 nm/20 excitation filter and a 585 nm/nm emissionfilter. Excited with a xenon arc lamp, the fluorescence emission ofBodipy-493 can only be detected in channel 1. Bodipy-564 emission can bedetected in channel 2. Bodipy-R6G can be detected in both channels. Asexpected, the scatter plot (FIG. 11) of the intensity in the twochannels of the beads clearly indicates three classes. Within eachclass, the beads have the same relative intensity ratio (correspondingto the same unit vector) with a certain uncertainties.

Similar design and data analysis procedure can be used for other dyesand detection channels.

1. A method of decoding an array so as to determine the location of acapture probe that has been randomly distributed on the array, saidmethod comprising the steps of: a) providing an array comprising aplurality of distinct capture probes, wherein said capture probes arerandomly distributed on said array; b) performing a hybridization stepin which said capture probes are hybridized with a plurality of sets ofdecoder probes, wherein each of said sets of decoder probes comprises adifferent label, each different label producing a different signal; c)detecting the signal produced by the hybridization step at a location onsaid array; d) performing a dehybridization step; e) performing anadditional hybridization step in which said capture probes arehybridized with a plurality of sets of decoder probes, wherein each ofsaid sets of decoder probes is different from said sets of decoderprobes used in the previous hybridization step, and wherein each of saidsets of decoder probes comprises a different label, each different labelproducing a different signal; f) detecting the signal produced by theadditional hybridization step at said location; g) repeating steps d-ffor a number of stages sufficient to generate a code comprising thesequence of signals detected at said location, said code indicative ofthe capture probe present at said location; and h) decoding said arraywith said code.
 2. The method of claim 1, wherein said array comprisesglass or plastic.
 3. The method of claim 1, wherein said array comprisesa fiber optic bundle.
 4. The method of claim 1, wherein said arraycomprises a flat planar surface.
 5. The method of claim 4, wherein saidflat planar surface comprises a depression comprising one or moreparticles.
 6. The method of claim 5, wherein said capture probes areattached to said one or more particles.
 7. The method of claim 6,wherein said one or more particles comprise microspheres.
 8. The methodof claim 6, wherein said one or more particles comprise beads.
 9. Themethod of claim 4, wherein said flat planar surface comprises at leastone well having a microsphere deposited therein.
 10. The method of claim1, wherein said capture probes and said decoder probes comprise nucleicacids.
 11. The method of claim 10, wherein said nucleic acids compriseDNA.
 12. The method of claim 1, wherein said labels comprise fluorescentlabels.